TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for monitoring of coagulation status and haemostasis in the perioperative setting. The invention also relates to an apparatus for applying said method, and to a cartridge applicable in said method, said cartridge being a flow cell. Furthermore, the invention relates to the use of said method, said apparatus and said cartridge for assessing coagulation status or thrombus formation in a whole blood sample. The invention also relates to comparing test results obtained with the method of the invention with test results in a database. In addition, the invention relates to a kit of parts comprising disposables required for applying said method of the invention with the apparatus of the invention.

BACKGROUND OF THE INVENTION

Practitioners, and especially surgeons and their operation team, are frequently confronted with patients suffering from mild to severe perioperative and postoperative blood loss or thrombosis, often under acute conditions and in the operation theater. These situations are for example typically familiar to surgeons performing cardiac surgery, vascular surgery and cardiopulmonary bypass surgery [van de Kerkhof & Herold, 2012; Bosch et al., 2014].

Blood loss in the operative context requires acute and adequate intervention, most preferably within a time-span of minutes. The underlying cause of the blood loss is divers and available therapies that could be applied are equally divers. Moreover, application of several therapies is diversified with regard to the underlying cause of the blood loss and with regard to the coagulation status of the bleeding patient.

Decisions as to which therapy should be selected for and applied to the patient suffering from the blood loss in the operation theater, are often made without implying results of clinical diagnostic tests in the decision making process [van de Kerkhof & Herold, 2012]. Currently available tests do not provide sufficient information for a balanced judgement as to which therapy should be selected for the bleeding patient or the patient with thrombosis [Tynngard et al, 2015]. Importantly, tests to monitor haemostasis adequately and reliably during and after surgery are currently either lacking, or do not have enough association with clinical bleeding or thrombosis, or even both [Bosch et al., 2014; Westein et al., 2012]. Moreover, the available tests do not provide results in a time- span shortly enough to wait for, before treatment of the patient can be started [van de Kerkhof & Herold, 2012; Bosch et al., 2014]. Added to these shortcomings of currently available tests are further shortcomings related to the requirement of sample preparation (of a too large volume of test sample, i.e. blood) before certain tests can be started, which is time-costly, and the required level of skills for executing the measurement and subsequently for analyzing and interpreting the data [Bosch et al., 2014].

For example, light-transmission platelet aggregation requires a relative large volume of platelet-rich plasma, and this assay takes a relatively long time, whereas it is doubted whether the assay results provide information relevant to the blood coagulation status in the bleeding patient. For example, routine coagulation tests require 20-30 minutes before test results become available; a time-span that is too long to wait for while the bleeding patient requires acute treatment.

Examples of nowadays clinical tests used in the perioperative setting are platelet aggregation tests such as the PFA-100 platelet function analysis with citrated whole blood, and viscoelastic whole blood clot formation tests, such as the ROTEM clot formation test. Although the PFA-100 test implies whole blood under shear conditions, it has been concluded that the test is not applicable for excessively bleeding patients.

Furthermore, sensitivity towards effects of anti-platelet therapy is insufficient. The PFA- 100 test has been judged as being not of importance in directing transfusion therapy strategy when excessive bleeding occurs after cardiac surgery [Forestier et al., 2002]. The ROTEM test is not able to detect impairment in platelet function induced by anti-platelet agents. Moreover, the ROTEM test, run under static conditions, therefore under non- physiological conditions by definition, requires about 23 minutes for delivering test results.

In many occasions one or several of these aforementioned tests are supplemented with routine clinical diagnostics tests, including activated partial thromboplastin time (aPTT), prothrombin time (PT) and platelet count for assessment of coagulation activity. However, these tests are too time consuming for being of relevance under the time- pressured circumstances as described. Moreover, platelet activity is not adequately assessed properly by these tests.

For at least 33% of the patients presented in the perioperative setting it is unknown whether or not they suffer from an increased risk for bleeding. The combination of tests as listed above provides inconclusive insights on coagulation status.

For already several decades, flow perfusion chamber technology bears the promising potential to overcome these above mentioned limitations of nowadays tests to assess haemostatic parameters in the clinic, for example in the perioperative setting related to bleeding patients or patients suffering from thrombosis. Flow perfusion chamber technology allows for assessment of platelet function and coagulation status at the same time, under conditions of (patho)physiological shear rates present in the circulation. Flow perfusion measurements provide information on a panel of parameters, such as platelet adhesion, platelet activation, thrombus growth, fibrin formation, coagulation, at selected venous or arterial shear rates. Platelet adhesive activity and thrombotic tendency of the blood of a patient can be analyzed while using various platelet-adhesive substrates as an activating surface. Such test results would be of tremendous value for the clinician confronted with the bleeding patient. Insight in the platelet adhesive activity and thrombotic tendency, complemented with the historical data on the patient and knowledge on current therapy regimens (e.g. aspirine therapy, anti-thrombotics, anti-cancer chemotherapy, eic), would contribute to a large extent to the decision making process of selection the potentially most beneficial therapy to stop bleeding or relief the patient from occlusions in the circulation.

The international application WO2006065739 provides an example of flow perfusion chamber technology meeting some though not all above mentioned limitations of haemostasis tests, and describes a device for aggregating, imaging and analyzing thrombi. An instrument is described comprising a plurality of channels which are partially or fully coated with a material that induces blood cell aggregation, and an imaging assembly and an analyzer for quantifying at least one characteristic of the aggregation. According to WO2006065739, the device is aimed at providing image data in less than 30 minutes, related to for example platelet adhesion and aggregation parameters for an individual. A video of thrombus formation is provided. For assessing brightfield

transmission signal, WO2006065739 provides a first analytical system, whereas for assessing fluorescence signal, WO2006065739 provides a second analytical system, thereby limiting the number of output parameters that can be obtained with a single measurements.

A different example of a flow perfusion chamber technology is provided in international patent application WO2015102726. A blood coagulation monitoring technology is described which takes into account shear stress and which requires minimal sample preparation and operator training. In WO2015102726, changes in pressure in micro-channels is recorded upon formation of a thrombus on a substrate, from blood.

In international patent application WO2010102335, yet another microfluidics device is presented, which provides real-time monitoring of platelet aggregation of a biological sample. The device of WO2010102335 comprises a flow cell comprising a flow channel with a protrusion. A biological sample comprising platelets flows through the flow channel and platelets are activated and aggregate once having contacted the protrusion while flowing. The platelet aggregation is assessed e.g. by optical detection means.

In patent application EP2040073, an alternative microfluidic device is described, for determining clotting time in a fluid medium such as blood. In the device, the blood flows through a first flow channel of a flow cell, comprising a region containing a reagent that reacts with the blood. In the same device, the blood flows through a second flow channel of a flow cell, not comprising a region containing a reagent that reacts with the blood. The measured difference in blood flow over time between the channels is a measure for the clotting of blood in the flow channel comprising the reagent.

Total thrombus-formation analysis system (T-TAS) is a flow perfusion system for quantitative assessment of thrombus formation under flow conditions. The system either provides information on thrombus growth, or, in a separate measurement, on platelet adhesion and aggregation. Test results are obtained either upon applying a surface of collagen in the T-TAS, or a surface of collagen with thromboplastin. Measurements with blood relate to pressure differences in occluding capillaries comprising the coated surface, on which e.g. activated platelets accumulate. It has been suggested that the T-TAS technique may perhaps complement viscoelastic whole blood clot formation tests in the clinic, such as the ROTEM clot formation test, although T-TAS still needs to be validated [Schott & Johansson, 2013].

Although flow perfusion chamber technology is a proven tool for application in research laboratories, suitable to derive information on e.g. platelet function and the influence of anti-thrombotic therapy on haemostasis status with prospective value, up till now the technology has been unable to repay expectations regarding beneficial implementation of the technology in the clinic [Westein et al., 2012; de Witt et al, 2014], e.g. during perioperative patient care. Entrance of the application of the technology in the clinic is severely hampered by a lack of standardization in chamber construction and in the use of the test method. The requirements of complex microscopic imaging analysis when applying the technology demands for highly trained personnel. In addition, the required volume of blood for running tests with the technology can be relatively large. Attempts to contribute to standardization and optimization of the flow perfusion chamber technology are numerous and regularly published [Westein et al., 2012; de Witt et al., 2014; Zwaginga et al., 2006; Roest et al., 201 1 ; Van Kruchten et al., 2012; Schott & Johansson, 2013]. However, none of these publications or aforementioned patent applications provide for adequate solutions yet, to the combination of drawbacks and shortcomings coming with the flow perfusion chamber technology, when use in the clinic is intended, e.g. in the perioperative setting involving patients with thrombosis or

(severely, acute) bleeding patients. Those skilled in the art repetitively acknowledge the need to overcome quite a number of hurdles before the flow perfusion chamber technology is ready for introduction into clinics [Schott & Johansson, 2013], e.g. in the perioperative setting during surgery. Hurdles related to for example the type of flow cell used in the technology, coating of surfaces, collection and storage of blood before start of the measurements, recording of digital images, methods of image analysis.

Thus, there is a strong and long-felt need for a sufficiently quick test for

qualitatively assessing coagulation status and haemostasis that provides reliable data associated with the perioperative and postoperative, often acute and severe blood loss or thrombosis, and that directs the practitioner to the most relevant therapy for the patient in need in a sufficiently short time-frame. In addition, the quick test should demand a minimal level of skills relating to operating the test and a minimal level of skills relating to analyzing and interpreting the data. Moreover, the test should be applicable for test samples without laborious sample preparation required, e.g. applicable for small volumes of freshly drawn ((non-)anti-coagulated) whole blood.

SUMMARY OF THE INVENTION

The inventors have developed a method for assessing coagulation status and

haemostasis status of a patient meeting the aforementioned desiderata.

A first aspect of the present invention therefore relates to an automated method for optically measuring complex formation under flow at physiological shear rate between a mobile binding partner and an immobilized binding partner, comprising the steps of:

a) providing a flow cell 4, 4', 4" comprising at least one flow channel 43, which flow channel comprises at least one reaction zone 45 located at a portion of the flow channel that is transparent for light, and wherein said reaction zone comprises an immobilized binding partner;

b) feeding an aqueous sample comprising the mobile binding partner to the reaction zone of the flow channel and allowing said mobile binding partner to form under flow of the aqueous sample a complex with the immobilized binding partner at physiological shear rate;

c) determining said complex formation by means of an light imaging assembly 50, wherein the light imaging assembly comprises a light source 30 and a light sensitive sensor 37, wherein the light sensitive sensor records in a time- lapse manner transmitted light signal from the light source 30 through the reaction zone; and

d) real-time processing and displaying the processed signal.

Preferably, the invention provides said method further comprising step aa) following step a):

aa) mixing the aqueous sample comprising the mobile binding partner with a light emitting label and allowing said label to bind to the mobile binding partner, such that a labeled mobile binding partner is formed,

and further comprising a step cc) preceding step d):

cc) further determining said complex formation by means of said light imaging assembly 50, wherein the light imaging assembly comprises a further light source

33,

wherein the light sensitive sensor records in a time-lapse manner light signal emitted by

the light emitting label bound to the mobile binding partner in the complex in the reaction zone upon irradiation of the complex by the light source 33,

wherein the light emitting label is selected from a fluorescent dye, a fluorescently labeled binding moiety, or combinations thereof.

The invention offers the advantage that it has now reduced to practice a method to retrieve information within a short time-frame most relevant to haemostasis status of a patient, whereas a minimal level of skills and intervention is required for retrieving said information.

A second aspect of the invention is an automated analytical system, comprising: (i) an imaging assembly (50) comprising seen along the direction of an optical path:

a) at least one light source (30) for transmission recordings;

b) at least one microscope objective lens (31 );

c) a high-reflectance mirror (32);

d) a semi-transparent mirror (34);

e) at least one wavelength filter (35);

f) at least one imaging lens (36);

g) an imaging sensor (37);

and further comprising

h) at least one light source (33) for fluorescence recordings; i) as well as;a socket 19 comprising an xyz stage controller 10 and a thermostat 9, for receiving a flow-cell holder 20;

j) a flow-cell holder 20,

(ii) a fluidic system 60, 60', 60" for feeding an aqueous sample comprising a

mobile binding partner to the flow channel 43 of a flow cell 4, 4', 4" comprising: a) at least one high-precision pump 1 , 1 ', connected with

b) at least one sample holder 2, 2', 13, the sample holder connected with c) a flow-through pump 5, 15, connected with

d) a multi-way valve 6, 16, connected with

e) at least one reservoir 11 , 17 comprising an aqueous solution A-C, D-F, wherein the aqueous solution is selected from Ca ²⁺ /Mg ²⁺ solution, physiological salt solution, buffer solution, wash buffer, rinse buffer, solution comprising a fluorescent label,

(iii) a flow cell 4, 4', 4" comprising at least one flow channel 43, 43' in connection with an inlet 42, 42' and an outlet 44, 44', wherein the flow channel comprises at least one reaction zone 45.

The automated analytical system of the invention is particularly suitable for carrying out the method according to the invention.

A third aspect of the invention is a flow cell 4, 4' for use in the automated analytical system according to the invention, comprising at least one flow channel 43, preferably two flow channels 43, 43', said flow channel comprising at least one reaction zone 45 located at a portion of the flow cell that is transparent for light, and said reaction zone comprising an immobilized binding partner, and said flow channel having an inlet 42, 42' positioned at an angle of 5-90° relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the at least one reaction zone in the flow channel, preferably at an angle of 10° to 20°, more preferably 1 1 ° to 12°, most preferably about 1 1 °, and an outlet 44 positioned at about the same angle as the angle of the inlet relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the reaction zone, and having a cross-sectional area of 0.075 mm ² to 0.30 mm ² , preferably with a width of about 2 mm or about 3 mm and a height of about 50 micrometer, with the reaction zone having a surface area of 1 .25 mm ² to 15 mm ² , preferably with a longitudinal dimension (length) of 0.8 mm to 3 mm and a transversal dimension (width) of 2 mm to 3 mm.

The flow cell of the invention is particularly suitable for use in the method according to the invention. A fourth aspect of the invention is the use of the automated analytical system according to the invention and a flow cell according to the invention for measuring haemostasis with the method of the invention by measuring at least one parameter selected from platelet deposition (thrombus surface area), thrombus build up, number of thrombi per surface area of immobilized binding partner, multilayer, P-selectin expression, phosphatidylserine exposure, fibrinogen binding, static platelet adhesion under non- coagulating conditions, and/or by measuring platelet deposition (thrombus surface area), thrombus build-up, time-to-fibrin formation, fibrin formation under coagulating conditions, and/or by measuring fibrinolysis under coagulating conditions, platelet-fibrin interaction, platelet aggregation, platelet adhesion, annexin A5 binding, granule secretion, formation of thrombin, platelet accumulation, thrombus volume, thrombus stability, immobilized platelet contraction, stenosis, granule secretion, clotting time, maximum lysis, platelet- based coagulation, or combinations thereof, preferably selected from platelet activation, platelet adhesion, thrombus formation, platelet-dependent fibrin formation under coagulating conditions, platelet-based coagulation, wherein the aqueous sample is whole blood.

A fifth aspect of the invention is a kit of parts comprising:

a) a flow cell 4, 4', 4" according to the invention for use in the automated analytical system according to the invention;

b) at least one holder with a further solution, selected from Ca ²⁺ /Mg ²⁺ solution,

physiological salt solution, buffer solution, wash buffer, rinse buffer, solution comprising a fluorescent label;

c) optionally a holder with at least one light emitting label according to the invention; and

d) a sample holder 2, wherein the sample holder is a syringe for blood draw, said syringe pre-filled with citrate and optionally pre-filled with at least one light emitting label according to the invention and with PPACK,

for use in a method according to the invention. DEFINITIONS

The term "time-lapse recording" as used herein has its normal scientific meaning and refers to time-lapse imaging with a digital camera, wherein images are captured at predetermined regular time intervals, i.e. the capture rate.

The term "coagulating condition" as used herein has its normal scientific meaning and refers to a fluid e.g. whole blood, that is kept in a state that allows for coagulation. For example, a coagulating condition for a whole blood sample is a volume of whole blood mixed initially with buffer solution comprising for example citrate, and then subsequently mixed with a solution comprising calcium cations.

The term "non-coagulating condition" as used herein has its normal scientific meaning and refers to a fluid e.g. whole blood, that is kept in a state that prevents coagulation. For example, an anti-coagulating condition for a whole blood sample is a volume of whole blood mixed with citrate, or citrate and PPACK.

The term "automated" as used herein has its normal scientific meaning and refers to a method or process that does not require any intervention by a (skilled) person up to the moment that a test resulted is provided by the method or process, once a test sample has been subjected to the method or process by the person.

The term "real-time", e.g. in 'real-time processing', as used herein has its normal scientific meaning and refers to the almost instantaneous processing of recorded signal data and subsequent storage and display of the processed signal data. In the context of the invention, "real-time", e.g. in 'real-time processing', results in the almost instantaneous availability of test results of the method of the invention, coinciding with the run-time of the complex formation under flow and the time-lapse recording of signal data.

DESCRIPTION OF THE FIGURES

Figure 1. A. A flow cell 4 with two flow channels 43 and 43' according to the invention

(side view / top view). Flow-cell 4: Top part 40; Bottom part 41 ; Inlet 42, 42'; flow-channels 43, 43'; Outlet 44, 44'; multiple reaction zones 45 comprising immobilized binding partner. Remark: for clarity, the top part 40 and the bottom part 41 are depicted with a spacing in between, indicated with the dashed lines. In the flow cell 4 according to the invention, the top part 40 and the bottom part 41 are in intimate contact (e.g. by means of sealing, gluing, pressing, etc.) according to the invention and when part of the automated analytical system of the invention. B. A flow cell 4' with a single flow channel 43 according to the invention. C. A flow cell 4" according to the invention, further comprising a sample holder 2'.

Figure 2. Drawings schematically showing an imaging assembly 50, as part of an automated analytical system according to the invention. The imaging assembly 50 of an automated analytical system according to the invention comprises, seen in the direction of an optical path defined by light traveling through the imaging assembly 50: Top light source 30 for trans-illumination; Flow cell 4' (or 4, 4"); Microscope objective lens 31 ; Mirror 32; Bottom light sources 33; Semi-transparent mirror 34; Wavelength filter 35; Imaging lens 36; Imaging sensor 37. In A., transmission is measured and signal data is time-lapse recorded upon illuminating the flow-cell 4' from above, with light source 30. In B. the fluorescence mode for measuring emitted light from a label which fluoresces is outlined. Here, the flow cell 4' is irradiated by the bottom light-source 33. In C, the socket 19 is depicted with thermostat 9 and xyz stage controller 10, and the flow-cell holder 20 for receiving a flow cell. The socket 19 intimately receives the flow-cell holder, for efficient heat transfer from the socket to the flow cell (heating the flow cell) or vice versa (cooling of the flow cell). The socket with the received flow cell holder comprising the flow cell is also depicted in A. and B.

Figure 3. A fluidic system 60 according to the invention, as part of the automated analytical system of the invention. For clarity reasons, the imaging assembly 50 of the automated analytical system is not depicted (see Figure 2 for the relative orientation of the imaging assembly with regard to a flow cell 4'). A. A schematic drawing of fluidic system 60 of the invention is outlined, wherein the fluidic system comprises a flow cell 4' having a single flow channel 43. Precision (syringe) pump 1 ; Sample holder 2, here a container, e.g. a syringe, for an aqueous sample, e.g. a whole blood sample, optionally mixed with blood mixable fluorescent dye(s); T-connector inlet connector 3, with an inlet 3' in connection with the outlet of the sample holder 2 and with an inlet 3" in connection with the outlet 5' of a flow-through micro pump 5 (see below); flow cell 4' with a single channel 43; flow through micro pump 5 having an outlet 5' connected to the T-connector inlet connector inlet 3", and having an inlet 5" in connection with the outlet 6' of a three-way selector valve 6 (see below); three-way selector valve 6, having an outlet 6' in connection with the inlet 5" of the pump 5, and having three inlets 6" in connection with three reservoirs 11 containing further aqueous solutions A, B, and C; outlet connector 7 in connection with the flow channel 43 of the flow cell 4; optionally, a waste container 8 in connection with the outlet connector 7; optionally a thermostat 9 for keeping at least the flow cell 4 at a predetermined temperature during complex formation and data collection; an xyz stage controller 10. B. A schematic drawing of an alternative fluidic system

60'according to the invention is outlined, wherein the fluidic system comprises a flow cell 4 having two flow channels 43, 43'. For elements 1 -3 and 5-11 of the fluidic system, see Figure 3A. Basically, the two-channel flow cell in Figure 3B comprises a further set of pumps, valves, etc. Precision (syringe) pump 12; Sample holder 13, here a container, e.g. a syringe, for an aqueous sample, e.g. a whole blood sample, optionally mixed with blood mixable fluorescent dye(s); T-connector inlet connector 14, with an inlet 14' (not shown) in connection with the outlet of the sample holder 13 and with an inlet 14" (not shown) in connection with the outlet 15' (not shown) of a flow-through micro pump 15 (see below); flow chamber 4 with two channels 43, 43'; flow through micro pump 15 having an outlet 15' (not shown) connected to the T-connector inlet connector inlet 14" (not shown), and having an inlet 15" (not shown) in connection with the outlet 16' (not shown) of a three- way selector valve 16 (see below); three-way selector valve 16, having an outlet 16' (not shown) in connection with the inlet 15" (not shown) of the pump 15, and having three inlets 16" (not shown) in connection with three reservoirs 17 containing further aqueous solutions D, E, and F; outlet connector 7 in connection with a first flow channel 43 of the flow cell 4; outlet connector 18 in connection with a second flow channel 43' of the flow cell 4; optionally, a waste container 8 in connection with the outlet connector 7 and in connection with the outlet connector 18. Remark: all inlets/outlets 14', 14", 15', 15", 16', 16" are not shown and are essentially similar to the inlets/outlets in Figure 3A, and as depicted with labels 3', 3", 5', 5", 6', 6", respectively. Figure 3C provides the schematic drawing of another embodiment of the invention, with yet an alternative fluidic system 60" according to the invention, wherein the fluidic system comprises a flow cell 4" having a single flow channel 43 and a sample holder 2'. The fluidic system comprises a precision (syringe) pump 1 '. The micropump 5 is now connected to the inlet 42 of the flow cell, via connector 3a.

Figure 4. Effect of storage of immobilized binding partner on reaction zones 45 under freezing conditions, regarding the extent of complex formation with platelets as the mobile binding partner in whole blood as the aqueous sample.

Figure 5. The effect of a tyrosine kinase inhibitor (TKI; anti-cancer drug) on thrombus formation. The effect of TKI in a whole blood sample is compared with a whole blood sample comprising vehicle only.

Figure 6. Test results of time-lapse recording of fluorescent signal from fluorescent labels bound to platelets, fibrinogen, phosphatidylserine as mobile binding partners in the complex, showing thrombus formation on collagen I +/- tissue factor as the immobilized binding partners, as measured with the method of the invention using an automated analytical system according to the invention.

Figure 7. Fibrin formation on an immobilized binding partner, with whole blood of a patient with hemophilia B as the source of fibrinogen/fibrin, in comparison to fibrin formation with whole blood of a healthy control.

Figure 8. Figures A-C show three different embodiments of the invention regarding time- lapse recording of transmitted light and fluorescence. In A., time-lapse recordings are performed at a single reaction zone with a flow cell comprising a single flow channel with a single reaction zone. Time-lapse recordings of brightfield transmitted light and fluorescence at three different wavelengths is sequentially alternated. First, for a time window, transmittance is recorded and analyzed. While signal is analyzed, the filter wheel repositions to a filter for a second wavelength. During recording of the first fluorescence signal, processed transmitted light data is stored and displayed. Subsequently, the first fluorescence data is processed while the filter wheel repositions to a third wavelength, for a second fluorescence recording etc., In Figure 1 B., a two-channel flow cell is used.

Again, recordings of brightfield transmitted light and fluorescence at three different wavelengths is sequentially alternated, now for both flow channels. For this, not only the filter wheel repositions between recordings, as in A., but in addition the xyz stage controller alternately moves the reaction zone in flow channel 43 and 43' in the beams of the top- and bottom light-source, once transmittance and three fluorescent signals are recorded at a first reaction zone. Figure C. provides an example of a scheme for time- lapse recording of transmitted data only. Now, within the same time span as in A. or B., more datapoints (images) are recorded. Total time from first to last recording is the same for A.-C, and is about 4 minutes or about 6 minutes. Further details about time lapse recording of images is provided in the detailed description of the invention. In C, time- lapse recordings are performed at a single reaction zone with a flow cell comprising a single flow channel with a single reaction zone. Time-lapse recordings of brightfield transmitted light is executed, only. For a time window, transmittance is recorded and analyzed. Signal is analyzed and processed transmitted light data is stored and displayed, before a second time-lapse recording of brightfield transmitted light is executed, etc., up till 4 minutes or six minutes of complex formation are completed. DETAILED DESCRIPTION OF THE INVENTION

It is a goal of the disclosure to address the above-indicated limitations related to the too long time-to-test results of current test methods, which test results should be sufficiently and reliably associated with the therapy to be selected for the (acute, severe) bleeding patient in need of the therapy, wherein applying said test and analyzing said test results should not require a significant level of skills from the test operator. Furthermore, it is a goal of the disclosure to address the limitation related to the required amount of test sample, i.e. blood of the patient, and related to test sample processing conditions in the test methods, which test method should closely mimic physiological circumstances.

The inventors now surprisingly found a method that addresses the aforementioned limitations. Therefore, a first aspect of the present invention relates to an automated method for optically measuring complex formation under flow at physiological shear rate between a mobile binding partner and an immobilized binding partner, comprising the steps of:

a) providing a flow cell 4, 4', 4" comprising at least one flow channel 43, which flow channel comprises at least one reaction zone 45 located at a portion of the flow channel that is transparent for light, and wherein said reaction zone comprises an immobilized binding partner;

b) feeding an aqueous sample comprising the mobile binding partner to the reaction zone of the flow channel and allowing said mobile binding partner to form under flow of the aqueous sample a complex with the immobilized binding partner at physiological shear rate;

c) determining said complex formation by means of an light imaging assembly 50, wherein the light imaging assembly comprises a light source 30 and a light sensitive sensor 37, wherein the light sensitive sensor records in a time- lapse manner transmitted light signal from the light source 30 through the reaction zone; and

d) real-time processing and displaying the processed signal.

The inventors developed a method that provides for a sufficiently short flow time, preferably about 4 minutes or about 6 minutes, required to allow for sufficient formation of a complex between the mobile binding partner and the immobilized binding partner.

'Sufficient' here refers to the formation of complex that reflects the physiological situation when the complex would have formed in the body, such that meaningful information can be retrieved from the method, i.e. information relating to complex formation between the binding partners in the body. Importantly, the inventors were able to combine the sufficiently short flow time, i.e. short enough for e.g. a surgeon waiting for test results directing the decision-making process of selecting a therapy for his (acute, severe) bleeding patient, with flow conditions that carefully mimic the conditions of flow and physiological shear rate as present in the circulation of the body. This aspect contributes to a large extent to the possibility to translate test results obtained with the method of the invention to the (patho)physiological conditions in the body.

Most importantly, the inventors also found that despite the short flow time in the method of the invention, it is possible to record a sufficient amount of relevant imaging data of sufficient intensity and contrast and sufficient detail, in order to provide the required test results in a shortly enough time-frame, upon processing the recorded signal data and subsequently displaying processed data as numerical data and/or in graphical form. Of course, within the same time span, also recorded images can be displayed, if required. Surprisingly, in the method of the invention, by combining the short flow time with time-lapse imaging mode (time-lapse signal data recording) at a carefully selected capture rate, the inventors found that this combination now allows for real-time processing of a sufficiently large set of recorded data signal, providing the required test results in a shortly enough time-frame at sufficient level of quality and detail. As a result, the invention addresses the current limitations as listed above by providing the relevant test results within a meaningful short time-frame. By combining flow perfusion technique and time- lapse image recording technique and data processing technique that addresses these limitations, the inventors were also able to automate the whole process from test sample introduction to delivery of the test results as numerical data and/or in graphics. Herewith, the method of the invention not only provides for a sufficiently fast process for assessing complex formation, including data acquisition and data processing, but the method of the invention also only requires a minimal level of skills.

Thus, with the method of the invention the inventors managed to align and shorten the time lines required for

a) building up enough complex between a mobile binding partner and an immobilized binding partner, by means of flow at physiological shear rate;

b) acquiring enough data of sufficient quality (sufficient signal-to-noise ratio), by

means of time-lapse image recording;

c) processing the recorded image data and providing at least one relevant output parameter as a test result by displaying numerical data and/or graphical data, in order to meet the time lines demanded by the clinical circumstances, e.g. a bleeding patient or a patient with thrombosis requiring a selected therapeutic intervention most dedicated to the unique coagulation status and haemostatic conditions of the patient.

With this method of the invention, for example when applying bright-field imaging (transmission mode), complex formation is for example visualized as accumulating aggregates of complexed mobile binding partner and immobilized binding partner in the reaction zone 45 surface area. This imaging technique is only one example of the many imaging techniques available today, and fit for the purpose of the method of the invention. For example, also images derived from fluorescence microscopy techniques are equally suitable for application in the method of the invention. Fluorescence imaging is a routine technique, and virtual any fluorescent label that can be coupled to a mobile binding partner is suitable for application in the method of the invention. In an embodiment of the invention, the method comprises a fluorescently labeled mobile binding partner. Alternatively, a mobile binding partner is provided with a fluorescent label by means of introducing a fluorescently labeled binding moiety, preferably a fluorescently labeled affinity molecule which binds to the mobile binding partner once exposed to it. Preferably, the fluorescent label or the fluorescently labeled affinity molecule with affinity for the mobile binding partner is provided in the aqueous sample comprising the mobile binding partner, before complex formation. Alternatively and equally preferred, said fluorescent label or said fluorescently labeled affinity molecule is bound to the mobile binding partner after the mobile binding partner and the immobilized binding partner have formed a complex. Optionally, the initial aqueous sample comprising the mobile binding partner has then first be replaced by a further aqueous solution, e.g. wash buffer, and the complex in the flow channel 43 has first been washed with said wash buffer, before the fluorescent label or the fluorescently labeled affinity molecule is bound to the mobile binding partner in the complex.

Preferably, the method of the invention further comprises a step aa) following step a):

aa) mixing the aqueous sample comprising the mobile binding partner with a light emitting label and allowing said label to bind to the mobile binding partner, such that a labeled mobile binding partner is formed,

and further comprising a step cc) preceding step d):

cc) further determining said complex formation by means of said light imaging assembly 50, wherein the light imaging assembly comprises a further light source 33, wherein the light sensitive sensor records in a time-lapse manner light signal emitted by the light emitting label bound to the mobile binding partner in the complex in the reaction zone upon irradiation of the complex by the light source 33, wherein the light emitting label is selected from a fluorescent dye, a

fluorescently labeled binding moiety, or combinations thereof.

Preferably, the fluorescent label is selected from a fluorescent dye selected from 3,3'-dihexyloxacarbocyanine iodide, DAPI, FITC, TRITC and CY5, the fluorescently labeled binding moiety is selected from a labeled antibody, antibody V domain, Fab fragment, ScFv, antibody chain, or combinations thereof.

Since the method of the invention is particularly suitable for assessing haemostatic conditions wherein the aqueous sample is whole blood or a blood-derivable aqueous sample, mobile binding partners that are particularly suitable for labeling with a light emitting label are phospatidylserine (PS), P-selectin, fibrinogen, fibrin, thrombin, allb33, von Willebrand factor (vWF), thrombospondin-1 , Factor V, Factor XII, Factor VIII, Factor IX, Factor X, and more preferably phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, or combinations thereof. For the skilled person it is immediately clear that several of these mobile binding partners are surface exposed molecules accessible at cells present in the blood sample or at cells exposed to the circulation, i.e. at the

(activated) platelet surface (e.g. phosphatidylserine), at the endothelial cell surface (e.g. P-selectin). Of course, also other mobile binding partners, e.g. such as those present in whole blood, involved in complexing with the immobilized binding partner are optionally labeled with a light emitting label, according to the invention.

According to the method of the invention, the labeled mobile binding partner is thus preferably selected from phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, von Willebrand factor, thrombospondin-1 , Factor V, Factor XII, Factor VIII, Factor IX, Factor X, or combinations thereof, more preferably phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, or combinations thereof.

Thus, even more preferably, in the method of the invention, the light emitting label is selected from a fluorescent dye selected from 3,3'-dihexyloxacarbocyanine iodide, DAPI, FITC, TRITC and CY5, a fluorescently labeled binding moiety selected from a labeled antibody, antibody V domain, Fab fragment, ScFv, antibody chain, or

combinations thereof, and the labeled mobile binding partner is selected from

phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, von Willebrand factor, thrombospondin-1 , Factor V, Factor XII, Factor VIII, Factor IX, Factor X, or combinations thereof, preferably phospatidylserine, P-selectin, fibrinogen, fibrin, thrombin, allb33, or combinations thereof.

Of course, time-lapse recording of transmitted light signal and/or of emitted light signal is performed according to the invention by time-lapse imaging during complex formation under flow, and/or at at least one time point at which complex has been formed and flow is (temporarily) reduced to 0 ml/hour, e.g. for an end-point recording and/or in a stop-flow set-up, known in the art. For end point recording, preferably the formed complex is first washed with an aqueous solution such as a wash buffer, before transmitted light signal and/or of emitted light signal is recorded.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium is exposed to the circulation. The platelet adhesion cascade takes place in the presence of shear flow. Flow-chamber, or perfusion-chamber, based assays to measure thrombus formation in vitro under conditions of shear flow are applied in the clinical research setting. These currently applied assays are laborious in nature. Applying the assays requires in-depth knowledge of the technology and biological mechanism underlying the assay and require a high level of skills in order to be able to properly operate such assays and interpret test results correctly. Most importantly, although the run-time of the assays, related to data acquisition, is in many occasions about 30 minutes or shorter, subsequent data processing and analysis is most often not only laborious, but also consumes further time in addition to the time required for data acquisition. That is to say, the sum of the time from the start of the data acquisition up to available test results is far over 10 minutes and in many occasions to up to an hour or even beyond. It is now due to the present inventors, that the method of the invention not only provides for an easy-to-use non-laborious manner of obtaining required test results on complex formation, without the need of an expert level of relevant skills, but also that the method of the invention concurrently provides for a manner fast enough to meet, e.g. the requirements demanded in the perioperative setting with regard to bleeding patients or patients with thrombosis in need of treatment on short notice. To facilitate for example therapy selection in the clinic, test results of the method of the invention are not only provided on short notice, but are optionally also related to a database comprising reference values. Such reference values preferably relate to previously acquired patient data, previously acquired test results with test samples reflecting a wide array of possible haemostatic conditions in the patient, etc. Of course, reference values optionally also relate to historical values obtained with test samples retrieved from the very same patient and measured with the method of the invention. This way, haemostatic condition of a patient is monitored over time with the method of the invention. For example, the pro- thrombotic status of a subject is assessed with the method according to the invention, by comparison of retrieved test results with a whole blood sample comprising at least platelets as a mobile binding partner, by comparison of the test results with reference control test results, i.e. reference values, stored in a database. Similarly, the bleeding status of a subject is assessed with the method according to the invention. The aqueous sample is preferably anti-coagulated citrated whole blood, the immobilized binding partner is typically collagen or collagen with tissue factor.

Thus, in the method according to the invention, optionally in a further step e) following step d) the processed data is related to a reference value, such that the amount and/or type and/or complex-forming capacity of mobile binding partner in the aqueous sample and/or in the complex can be determined, and/or such that the amount or type of formed complex or the rate of complex formation can be determined. As said, it was not until the inventors provided the method according to the invention, bearing an ingeniously and fine-tuned combination of features allowing fast complex formation under physiological conditions of flow and shear rate, fast acquisition of enough signal data of sufficient quality and intensity (low signal-to-noise ratio), and quick provision of test results by balancing time-lapse data acquisition with a data processing algorithm meeting the required tight time limit, that now can adequately address the limitations felt in the clinic. In order to resemble physiological conditions of flow and shear rate in the circulation of the body, flow and shear rate as applied in the method according to the invention, are carefully determined and selected. Shear rates occurring in the circulation of the body are between 0 s ^"1 , e.g. in an artery or vein at the downside of an occlusion, and about 50.000 s ^"1 , in e.g. a nearly occluded artery. In many occasions, these shear rates are between about 75 s ^"1 and 2.000 to 10.000 s ^"1 , or at about 150 s ^"1 for veins and at about 1.000 to 1 .600 s ^"1 for arteries. Therefore, preferably in the method according to the invention, shear rates are applied that fall within these mentioned physiological ranges. Particularly preferred in the method of the invention are shear rates between about 150 s ^"1 and about 1 .600 s ^"1 , preferably selected from about 150 s ^"1 , about 1.000 s ^"1 , about 1 .600 s ^{" 1} , therewith closely mimicking the physiological conditions in veins and in arteries of the body.

Thus, preferably, in the method according to the invention, the shear rate is 150 s ^"1 to 1 .600 s ^"1 , preferably selected from about 150 s ^"1 , about 1 .000 s ^"1 , 1 .600 s ^'

In addition to the shear rate, in the method of the invention the flow rate is optimized for meeting the requirements of desired shear rate and simultaneously for facilitating complex formation within a short time frame and with sufficiently high quality suitable for providing high quality test results with the method of the invention. Typically, in the method of the invention, flow of the aqueous sample comprising the mobile binding partner (or of a subsequently applied aqueous solution, e.g. the subsequent buffer; see above) is provided at between 0 ml/hour (when e.g. stop-flow is applied or when an end- point data acquisition is performed) and about 300 ml/hour, preferably the flow is at about 0.6 ml/hour to about 225 ml/hour, more preferably, the flow is about 0.675 to 10 ml/hour, most preferably about 7.2 ml/hour or about 0.675 ml/hour or about 4.5 ml/hour. When the aqueous sample is whole blood and when the flow is about 7.2 ml/hour, the shear rate is about 1 .600 s ^"1 , according to the invention. When the aqueous sample is whole blood and when the flow is about 0.675 ml/hour, the shear rate is about 150 s ^"1 , according to the invention. When the aqueous sample is whole blood and when the flow is about 4.5 ml/hour, the shear rate is about 1 .000 s ^"1 , according to the invention. Thus, preferably, in the method according to the invention, the flow is 0.6 to 225 ml/hour, more preferably 0.675 to 10 ml/hour, more preferably about 7.2 ml/hour, or about 0.675 ml/hour, or about 4.5 ml/hour.

According to the method of the invention, an ingenious balance is found between at one hand time-lapse recording of images suitable for capturing enough signal and enough detail of the complex at a selected capture rate, providing high-quality signal data for subsequent processing (i.e. with a shutter time of the imaging sensor 37 of about 10 ms to about 200 ms), and at the other hand creating a time-gap between consecutive steps of time-lapse data signal recording, which time gap is just long enough for allowing storage of acquired data, processing said data, storing and displaying processed data (numerical, graphical, image) and optionally relating the processed data (i.e. the test result) to a reference value. Importantly, according to the invention the imaging and subsequent storing, processing and displaying is in real time. That is to say, the complex formation is within 4 to 6 minutes, preferably in about 4 minutes or in about 6 minutes. Recording of images during complex formation and all subsequent image processing steps, eic, all together are also finished within these 4 to 6 minutes. The method of the invention can also provide test results within 30 seconds to 4 minutes, if complex formation is also established within these same 30 seconds to 4 minutes.

In the method of the invention, the time gap is a time interval of between 500 ms to 60 seconds between consecutive time-lapse recordings, preferably 600 ms to 30 seconds, preferably 600 ms to 6 seconds, most preferably about 800 ms to 990 ms, or about 1 second, or about 2 seconds, or about 6 seconds. Within this time gap, or time interval, recorded signal data (i.e. an image) is stored, processed, displayed and optionally compared with reference values from a database. In the method of the invention, time for each recording, i.e. the shutter time, is about 10 ms to about 200 ms for each image recorded.

Thus, preferably, in steps c) and cc) of the method according to the invention, signal is time-lapse recorded with a shutter time of the imaging sensor 37 of about 10 ms to about 200 ms, and with a time gap of 500 ms to 60 seconds, preferably 600 ms to 30 seconds, more preferably 800 ms to 990 ms.

Example 17 and Figures 8A-C provide embodiments of the invention, showing various routines for measuring complex formation by time lapse imaging and

simultaneously processing the images real-time.

When images are recorded of transmitted light and/or of at least one emitting fluorescent label, time lapse recordings and subsequent storing, processing, etc., are performed in consecutive order. Thus, for example, first transmittance is recorded for 10- 200 ms, and subsequently signal is stored, processed, etc. Then, the filter wheel is turned to a new position allowing recording of fluorescence of a first label which signal is stored, processed, etc. Then, the filter wheel is again turned to a new position allowing recording of fluorescence of a second label which signal is stored, processed, etc. Then, the filter wheel is turned to the starting position allowing again recording of transmitted light which signal is stored, processed, etc. These cycles of time lapse imaging followed by the time gap are repeated during the course of complex formation, thus preferably during about 4 to 6 minutes. In the method of the invention, at least time lapse signal (brightfield) of transmitted light is recorded, and preferably also time lapse signal of at least one fluorescent label is time lapse recorded, preferably of one, two, three or four, or more different fluorescent labels. Most preferably, transmittance is recorded and fluorescent signal at 1 to 4 wavelengths, relating to 1 to 4 different fluorescent labels is recorded. See Figure 8 for preferred embodiments of the invention, related to time lapse recordings of transmitted light and fluorescence, with a flow cell having a single flow channel or having two flow channels, for example.

As mentioned before, time is key in for example circumstances that require adequate and immediate intervention. One such circumstances is when a bleeding patient in the operation theater is in need of a therapy, which therapy is first to be selected from a plethora of available therapies and at least one most suitable therapy. The method of the invention, now, provides guidance for the process of selecting the at least one most adequate therapy for the (acute, severely) bleeding patient in need of said therapy.

Fortunately, the method of the invention provides this guidance by providing the relevant test results (numerical data, graphical data, if required supplemented with one or a few recorded images) within a time-frame short enough for application in the perioperative setting of the operation theater, e.g. within 4 to 6 minutes.

Thus, most preferably, the method according to the invention, provides the test results within 30 seconds to 30 minutes, preferably 2 minutes to 15 minutes, more preferably 3 minutes to 10 minutes, even more preferably 4 minutes to 6 minutes, most preferably at about 4 minutes or at about 6 minutes.

Thus, in the method of the invention, preferably steps a) to d) or steps a) to e) together take 30 seconds to 30 minutes, more preferably 2 minutes to 15 minutes, more preferably 3 minutes to 10 minutes, most preferably 4 minutes to 6 minutes, or about 4 minutes or about 6 minutes. Thus, most preferably, in the method according to the invention, the flow is 0.6 to 225 ml/hour, more preferably 0.675 to 10 ml/hour, more preferably about 7.2 ml/hour, or about 0.675 ml/hour or about 4.5 ml/hour, wherein the shear rate is 150 s ^"1 to 1.600 s ^"1 , preferably selected from about 150 s ^"1 , about 1 .000 s ^"1 , about 1 .600 s ^"1 , wherein in steps c) and cc) signal is time-lapse recorded and stored at time intervals of 600 ms to 60 seconds, preferably 600 ms to 30 seconds, most preferably about 600 ms, or about 1 second, or about 2 seconds, or about 6 seconds, and wherein steps a) to d) or steps a) to e) together take 30 seconds to 30 minutes, preferably 2 minutes to 15 minutes, more preferably 3 minutes to 10 minutes, even more preferably 4 minutes to 6 minutes, most preferably about 4 minutes or about 6 minutes.

Since regularly, many processes in life have a multi-factor dependency on several parameters, mimicking such a process by applying a single method which provides a single or a few test results, is often a difficult task. Fortunately, the method of the invention allows for assessing complex formation in at least one reaction zone and up to about 20 reaction zones 45, preferably 1 to 9 reaction zones 45, most preferably 3 to 5 reaction zones 45, in a single application run of the method. When applying a flow cell with two or multiple flow channels, with each flow channel comprising multiple reaction zones, even more parameters are assessable within a single measurement according to the method of the invention. This opens the way for applying the method of the invention in a mode wherein multiple parameters and/or multiple conditions that influence complex formation, are assessed simultaneously, within a single measurement. For example, a concentration series of immobilized binding partner divided over multiple reaction zones 45 is assessed with the method of the invention, and/or various individual immobilized binding partners are analyzed in separate reaction zones 45, and/or various combinations of different immobilized binding partners are analyzed in separate reaction zones 45, or combinations thereof. These modes of the method according to the invention optionally widens the window of test results that is provided.

In the method according to the invention, preferably the flow channel 43 comprises one reaction zone 45 with immobilized binding partner or comprises multiple reaction zones 45 with immobilized binding partner, preferably 1 to 20 reaction zones 45, more preferably 1 to 9 reaction zones 45, most preferably 3 to 5 reaction zones 45.

Thus, a method according to the invention is provided, wherein preferably the flow cell 4 comprises two or more reaction zones 45 comprising a concentration series of an immobilized binding partner. It is part of the invention that if multiple reaction zones 45 in the method of the invention are applied in the flow channel 43 in consecutive manner (See Figure 1 B for an exemplifying embodiment of the invention), as opposed to an equally suitable parallel arrangement of reaction zones 45, or combinations thereof, the immobilized binding partners are immobilized at the multiple reaction zones 45 in an order from relative low complex-forming capability to high complex forming ability, with respect to the direction of the flow. This way, aqueous sample entering the flow channel 43 first contacts

immobilized binding partner with relative low complex-forming capability such that mobile binding partner at first contacts such immobilized binding partner with relative low complex-forming capability. Subsequently, a next immobilized binding partner with relative higher complex-forming capability contacts the mobile binding partner, etc. For example, in the method of the invention, a first reaction zone 45 comprises the immobilized binding partner collagen and a second reaction zone 45 comprises immobilized binding partner tissue factor combined with collagen, wherein the aqueous sample is whole blood and the mobile binding partner is a platelet and/or fibrinogen and/or a blood coagulation factor, or combinations thereof.

When assessing complex formation with the method of the invention, of course proteins and peptides are suitable immobilized binding partners. In addition, and equally preferred, are cells of any type as immobilized binding partners in the method of the invention. Also combinations of proteins and peptides, and cells are optionally applied as immobilized binding partners in the method of the invention. Cells particularly suitable for application as immobilized binding partner in the method of the invention are cells that contact whole blood in the circulation of the body under physiological conditions or under pathophysiological conditions. In this regard, according to the invention, a preferred cell as immobilized binding partner is for example a platelet, a red blood cell, a vascular cell, a tissue cell, a macrophage, a T-cell, a B-cell, a NK-cell, a monocyte, a neutrophil, an endothelial cell, a muscle cell, a vascular smooth muscle cell, a fibroblast, or combinations thereof, more preferably an endothelial cell, a vascular smooth muscle cell, or a cell related to inflammation selected from a monocyte, a neutrophil, or combinations thereof.

Thus, a method according to the invention is provided, wherein the immobilized binding partner is selected from a single protein, a mixture of proteins, a single cell type, a mixture of cell types, a complex-inducing material, or a combination thereof.

In the method according to the invention, preferably the immobilized binding partner is selected from a single protein or a mixture of proteins and wherein the mobile binding partner is selected from a single protein, a mixture of proteins, a single cell type, a mixture of cell types, or a combination thereof.

Optionally, in a further preferred embodiment of the method of the invention, the immobilized binding partner comprises a material such as an artificial surface material, wherein said material activates blood platelets and induces complex formation and platelet aggregation. Examples of such materials applicable in the method of the invention are materials applied in stents, surface materials of any apparatus or part that contacts the circulation in the body, a negatively charged surface material, a mechanical valve, and the like. Preferred materials for use as an immobilized binding partner according to the invention are for example a polyurethane, a polyvinylchloride, a polymethylmethacrylate, or combinations thereof.

Now that the invention provides a fast and reliable method for assessing complex formation between a mobile binding partner and an immobilized binding partner under conditions of flow at physiological shear rate, the method of the invention is of course particularly suitable for assessing formation of complexes as is occurring in the circulation of the body, under (patho)physiological conditions. The immobilized binding partner is thus preferably selected from at least a molecule or at least a cell, or combinations thereof, that are also present in immobilized form in the circulation or that are exposed to the circulation under pathophysiological conditions.

Thus, in the method according to the invention, preferably the immobilized binding partner comprises at least one protein selected from laminin, proteoglycan, tissue factor (TF), fibrin, fibrinogen, fibronectin, vitronectin, osteopontin, collagen, collagen type I, collagen-derived peptide, collagen peptide mimetic, rhodocytin, von Willebrand factor, tissue thromboplastin, activated protein C, factor XII, P-selectin, annexin A5, integrin a2, integrin ανβ3, integrin α5β1 , CD36, integrin allbp3, glycoprotein VI, integrin α2β1 , glycoprotein Iba, CLEC-2, coagulation factors or combinations thereof, or a(n artificial) material that triggers a platelet activation pathway or that triggers a platelet inhibitory pathway, or combinations thereof, more preferably selected from collagen, von Willebrand factor, tissue thromboplastin, activated protein C, factor XII, laminin, fibrinogen, fibronectin, tissue factor, proteoglycan, or combinations thereof.

According to the invention, the aqueous sample is preferably selected from culture medium, blood serum, blood plasma, buffer solution or a cell suspension selected from whole blood, plasma, synovial fluid, cerebrospinal fluid, lymph, interstitial fluid, cells in culture medium, cells in buffer solution, or any dilutions thereof and/or any combinations thereof, more preferably, whole blood, and the mobile binding partner comprises a cell selected from a platelet, a red blood cell, a cell of the vascular lining, a human umbilical vein endothelial cell, a patient-derived blood outgrowth endothelial cell, a tissue cell, a macrophage, a T-cell, a B-cell, a NK-cell, a monocyte, a neutrophil, a leukocyte, a progenitor cell, an endothelial cell, a muscle cell, a vascular smooth muscle cell, a fibroblast, or a combination thereof, more preferably, a platelet, and even more preferably the aqueous sample is whole blood and the mobile binding partner is at least a platelet, preferably a platelet and at least a protein, such as fibrinogen and/or a blood coagulation factor.

When an aqueous sample is applied in the method of the invention, and the sample is a whole blood sample, the whole blood sample is typically an anti-coagulated whole blood sample according to the invention. According to the invention, at least one anti-coagulant is added to a whole blood sample, selected from D-Phenylalanyl-L-propyl- L-arginine chloromethylketone (PPACK), fragmin, hirudin, heparin, citrate, corn trypsin inhibitor (CTI) or other anticoagulants, or combinations thereof. Preferably, citrated whole blood samples are used as aqueous samples according to the invention ('coagulating' conditions). Also preferred as aqueous samples according to the invention are whole blood samples provided with citrate and PPACK at concentrations known in the art ('anti- coagulating' conditions).

When assessing haemostatic balance with a whole blood sample using the method according to the invention, valid results are retrieved with multiple reaction zones 45 comprising either collagen, or von Willebrand factor, or tissue factor, or combinations thereof. Importantly, coagulation status is retrieved with a whole blood sample under coagulating conditions, in the method of the invention.

Referring to the aforementioned limitations currently experienced in the clinic, under conditions of a bleeding patient in need of immediate help, any method to be applied that requires a substantial volume of blood from the patient is heavily undesired. Furthermore, due to the tight time constraints regarding the moment of occurrence of the (acute, severe) bleeding episode up to the start of a selected therapy, any required sample handling and preparation which takes (too much) time is also limiting the applicability of any test aimed at facilitating patient care. The method of the current invention, now, addresses both limitations adequately. First, the method of the invention is directly applicable with whole blood samples of the patient, obtained following routine practice in the operation theater, without the need of any sample preparation before the start of the method of the invention. Second, the method according to the invention only requires less than a milliliter of blood, preferably 200 microliter to 1 milliliter, most preferably about 500 microliter.

Thus, preferably, in the method according to the invention, the aqueous sample is a sample of 200 microliter to 1 ml, preferably about 500 microliter.

Thus, even more preferably, in the method according to the invention, the aqueous sample is 200 microliter to 1 ml whole blood, preferably about 500 microliter.

Equally preferable according to the invention, the aqueous sample is 200 microliter to 1 ml citrated whole blood provided with calcium and magnesium ions, preferably about 500 microliter ('coagulating' conditions).

Equally preferable according to the invention, the aqueous sample is 200 microliter to 1 ml citrated whole blood provided with calcium and magnesium ions and with PPACK, preferably about 500 microliter ('anti-coagulating' conditions).

In addition to the important parameters, i.e. flow and shear stress, that contribute to mimicking (patho)physiological conditions in the circulation of the body, also the temperature applied in the method of the invention contributes to the high degree of mimicking physiological conditions as seen with the method of the invention. Typically, the temperature during complex formation and during data acquisition is 4°C to 42°C, preferably 10°C to 39°C, most preferably ambient temperature to about 37°C. Especially when a whole blood sample, with/without citrate and/or PPACK, and with/without calcium ions and magnesium ions, is assessed as an aqueous sample comprising a mobile binding partner, with the method according to the invention, most preferably the temperature during complex formation is at ambient temperature to about 37°C.

Thus, in the method according to the invention, preferably the temperature during steps a)-d) or a)-e) of the method of the invention is 4°C - 42°C, preferably 10°C - 39°C, most preferably at ambient temperature to about 37°C.

Thus, even more preferably, in the method according to the invention, the aqueous sample is 200 microliter to 1 ml whole blood, preferably about 500 microliter, wherein the mobile binding partner is selected from a platelet, a blood coagulation factor or combinations thereof, and wherein the immobilized binding partner comprises at least one protein selected from laminin, proteoglycan, tissue factor, fibrin, fibrinogen, fibronectin, vitronectin, osteopontin, collagen, collagen type I, collagen-derived peptide, collagen peptide mimetic, rhodocytin, von Willebrand factor, tissue thromboplastin, activated protein C, factor XII, P-selectin, annexin A5, integrin a2, integrin ανβ3, integrin α5β1 , CD36, integrin allbp3, glycoprotein VI, integrin α2β1 , glycoprotein Iba, CLEC-2, coagulation factors or combinations thereof, or a(n artificial) material that triggers a platelet activation pathway or that triggers a platelet inhibitory pathway, or combinations thereof, preferably selected from collagen, von Willebrand factor, tissue thromboplastin, activated protein C, factor XII, laminin, fibrinogen, fibronectin, tissue factor, proteoglycan, or combinations thereof, and wherein the temperature during steps a)-d) or a)-e) is 4°C - 42°C, preferably 10°C - 39°C, most preferably at ambient temperature to about 37°C.

Preferably, in the method according to the invention the immobilized binding partner in the reaction zone 45 comprises collagen type I or collagen type I and tissue factor, wherein the mobile binding partner comprises a platelet, wherein the aqueous sample comprises anti-coagulated whole blood, wherein a further solution is a reaction mixture comprising an anti-coagulant, wherein the light emitting label moiety is 3,3'-dihexyloxacarbocyanine iodide, wherein the flow is 0.6 to 30 ml/hour, preferably 0,675 to 10 ml/hour, more preferably about 7.2 ml/hour, and wherein steps a) and onwards take 4 to 6 minutes. Preferably, the temperature during complex formation and during time-lapse recording of signal data is at about ambient temperature to about 37°C.

Preferably, the aqueous sample is citrated whole blood comprising PPACK, calcium ions and magnesium ions (non-coagulating conditions). Then, preferably, steps a) and onwards, up to d) or e) last about 4 minutes, according to the method of the invention. Equally preferred is when the aqueous sample is citrated whole blood comprising calcium ions and magnesium ions (coagulating conditions). Then, preferably, steps a) and onwards, up to step d) or step e) last about 6 minutes, according to the method of the invention.

The method of the invention has been proven to be adequately sensitive for detecting variations in platelet count in a whole blood sample. Also coagulation defects that influence coagulation status of a subject suffering from such defect, is adequately measured with the method according to the invention. Moreover, when whole blood samples from subjects who receive anti-platelet therapy or subjects who receive antithrombotic therapy, are subjected to the method of the invention, sensitive measurements reveal meaningful and informative test results.

Preferably, the method according to the invention provides the relevant information on the activation of platelets, formation of a thrombus, platelet-based coagulation with platelets of a patient. More preferably, the method according to the invention provides test results relating to the platelet aggregation status, platelet adhesion status and/or platelet- fibrin interaction status in a whole blood sample of a patient. The method according to the invention is typically suitable for providing parameter values regarding platelet aggregation, thrombus size, granule secretion, fibrinogen binding, clotting time and maximum lysis. These parameters provide guidance to for example the ones responsible for selecting at least one adequate therapy for a patient suffering from (severe, acute) bleeding or suffering from (acute) thrombosis, for example in the perioperative or postoperative context, for example during or after surgery in the operation theater.

A second aspect of the invention is an automated analytical system, comprising:

(i) an imaging assembly 50 comprising:

a) at least one light source 30 for transmission recordings;

b) at least one light source 33 for fluorescence recordings;

c) a semi-transparent mirror 34;

d) a high-reflectance mirror 32;

e) at least one wavelength filter 35;

f) at least one imaging lens 36;

g) an imaging sensor 37;

h) at least one microscope objective lens 31 ;

i) a socket 19 comprising an xyz stage controller 10 and a thermostat 9, for receiving a flow-cell holder 20;

j) a flow-cell holder 20,

(ii) a fluidic system 60, 60', 60" for feeding an aqueous sample comprising a mobile binding partner to the flow channel 43 of a flow cell 4, 4', 4" comprising a) at least one high-precision pump 1 , 1 ', connected with

b) at least one sample holder 2, 2', 13, the sample holder connected with c) a flow-through pump 5, 15, connected with

d) a multi-way valve 6, 16, connected with

e) at least one reservoir 11 , 17 comprising an aqueous solution A-C, D-F, wherein the aqueous solution is selected from Ca ²⁺ /Mg ²⁺ solution, physiological salt solution, buffer solution, wash buffer, rinse buffer, solution comprising a fluorescent label,

(iii) a flow cell 4, 4', 4" comprising at least one flow channel 43, 43' in connection with an inlet 42, 42' and an outlet 44, 44', wherein the flow channel comprises at least one reaction zone 45 with the reaction zone having a surface area of 1 .25 mm ² to 15 mm ² , preferably with a longitudinal dimension (length) of 0.8 mm to 3 mm and a transversal dimension (width) of 2 mm to 3 mm. The flow cell of the automated analytical system of the invention preferably has outer dimensions of about 24 mm x 60 mm x 5 mm (longitudinal width x transversal length x height), but other dimensions are equally preferred. Conventional glass cover slips are suitable for use as a bottom part 41 of a flow cell of the invention, comprising the at least one reaction zone. These conventional cover slips are 24 mm x 60 mm (longitudinal width x transversal length). Further embodiments of flow cells according to the invention are provided in Example 1 and Figure 1.

A preferred embodiment of a fluidic system of the automated analytical system of the invention is provided in Example 3 and Figure 3. The fluidic system of the invention preferably comprises said at least one reservoir 11 , 17 for containing a further aqueous solution, for example for the purpose of providing a wash buffer or a solution comprising a fluorescent label to formed complex. Said at least one reservoir also comprises a container comprising a solution comprising calcium ions and magnesium ions

(recalcification buffer). For several applications of the method according to the invention, using the automated analytical system of the invention, flushing the flow channel 43 with wash buffer once complex has been formed, is beneficial before a (time-lapse) recording of signal is applied, for example before an end-point recording is applied.

Preferably, the fluidic system of the invention comprises tubings, connectors, interior of pumps, etc., which are made of non-adhesive material. For example, connectors are preferably made of Teflon. When the aqueous sample is citrated whole blood and the mobile binding partner are platelets, use of Teflon surfaces in the fluidic system of the invention has the advantage that platelets contacting these surfaces are minimally pre-activated before being contacted with immobilized binding partner in the flow channel. Thus preferably also the inner lining of the flow channel is made of non- adhesive material. Preferably the inner lining of the flow channel is also made of non- activating material, when platelets and coagulation factors are concerned.

Light source 30 of the imaging assembly of the automated analytical system of the invention is preferably a diffuse white LED, suitable for brightfield transmission recordings. The LED is preferably a non-modulated LED or a pulsed LED. Light source 33 of the imaging assembly of the automated analytical system of the invention is preferably a diffuse RGB LED. Equally preferable is a plurality of fluorescence lamps, for example four fluorescence lamps. Preferable, the light source 33 is applicable for fluorescent excitation of dyes in the range of 390 nm, 465 nm, 488 nm, 549 nm and 647 nm. When separate lamps are applied as light source 33, a filter wheel 35 is part of the imaging assembly, said filter wheel preferably comprising for example filters: 1 ) dapi, long pass, > 435 nm; 2) DIOC (488 nm), 525 nm/50 nm; 3) Cy3 605 nm/70 nm; 4) Cy5 700 nm/75 nm.

Optionally, the automated analytical system of the invention comprises an imaging assembly according to the invention with a light source 30. Then, light source 33, mirror 34, filters 35 and lens 36 are not comprised by the imaging assembly. Such an automated analytical system is suitable for time lapse recording of transmitted light and provides test results suitable for application in the perioperative setting.

According to the invention, a preferred automated analytical system of the invention comprises a fluidic system which comprises filters 35 for filtering light of 2-6 wavelengths, preferably 5 wavelengths, more preferably 4 wavelengths emitted by the at least one light emitting label.

Of course, these filters 35, which are commonly known in the art, are typically suitable for selecting wavelengths applicable for measurements involving recording at least one emitted light according to the invention.

The xyz stage controller of the imaging assembly according to the invention is a conventional xyz stage controller known in the art. The xyz stage controller is preferably fixed to the socket which receives the flow cell holder comprising the flow cell. The xyz controller controls the focusing of the reaction zone (z-direction), and controls the positioning of a selected reaction zone in the area where light of light source 30 or 33 reaches the reaction zone and transmitted light or fluorescent signal from the complex in the reaction zone can reach lens 31 (xy positioning). The xyz stage controller may also be fixed to for example lens 31 in an alternative embodiment of the invention.

The flow cell holder is preferably a holder separate from the socket. Once a flow cell is received by the flow cell holder, it is easily mounted in the socket. The holder and socket have a close fit, allowing efficient thermal conduction between the thermostat mounted on the socket and the flow cell in the flow cell holder. Alternatively and also a preferred embodiment of the invention is a socket with a fixed flow cell holder. A preferred embodiment of a socket and a separate flow cell holder is provided in Figure 2 and Example 2.

The imaging assembly of the invention comprises a microscope objective lens.

The lens preferably magnifies 40x or 60x although other magnifications are also applicable in the automated analytical system of the invention. Combining the microscope objective lens with an imaging sensor makes the use of a conventional microscope superfluous, Preferably, the imaging sensor is a CCD sensor known in the art or a CMOS sensor known in the art. Preferred embodiments of microscope objective lenses and imaging sensors are provided in Figure 2 and Example 2. Now that no conventional microscope is required for time lapse recording of images of light transmitted through complex and/or of fluorescence emitted by label in the formed complex, according to the invention, a small and compact automated analytical system of the invention is provided. Furthermore, obeying a microscope has the further advantage that the risk for recording blurry images reduced with the use of the imaging assembly of the invention.

Preferably, the automated analytical system according to the invention comprises means 9, i.e. a thermostat to allow complex formation according to the invention at a temperature selected from 4°C - 42°C, preferably between 10°C - 39°C, most preferably at ambient temperature to about 37°C. Preferred is the ability to maintain the temperature at ambient temperature or at 37°C during complex formation.

See for preferred embodiments of an automated analytical system of the invention Figures 1 -3 and Examples 1 , 2, 3. The drawings and examples incorporated herein and accompanying this description of the invention constitute part of the specification. The drawings and examples exemplify and illustrate embodiments of the invention, and together with the description provided here above and below, serve to explain the features of the invention.

The automated analytical system of the invention is preferably small-size, e.g. with dimensions not exceeding about 40 cm x about 40 cm x about 40 cm (depth x width x height), preferably not exceeding about 30 cm x about 30 cm x about 30 cm (depth x width x height), more preferably the automated analytical system of the invention is an integrated and miniaturized system with dimensions of about 25 cm x about 25 cm x about 25 cm (depth x width x height). That is to say, the automated analytical system of the invention is preferably 'miniaturized'. Such a miniaturized system according to the invention bears the benefit of occupying a limited surface area of valuable space in e.g. the operation theater. In one preferred embodiment of the invention, the automated analytical system comprises a housing comprising the imaging assembly, the fluidic system and the flow cell according to the invention. In an even more preferred

embodiment of the invention, said housing has a dimension of about 30 cm x about 30 cm x about 30 cm (depth x width x height). In one preferred embodiment of the invention, the housing further comprises computer assisted means configured for manually starting the imaging assembly and the fluidic system, wherein the imaging assembly further comprises computer assisted automated means configured for time-lapse recording transmitted light and/or emitted light, and computer assisted automated means configured for real-time storing and processing time-lapse recorded data, and displaying said processed data, and wherein the fluidic system further comprises computer assisted automated means configured for driving the high-precision pump and the flow-through pump. In another preferred embodiment of the invention, said computer assisted means of the invention are not comprised in the housing comprising the automated analytical system, though are positioned proximate to said automated analytical system.

Thus, preferably, the automated analytical system according to the invention, further comprises computer assisted means configured for manually starting the imaging assembly and the fluidic system, wherein the imaging assembly further comprises computer assisted automated means configured for time-lapse recording transmitted light and/or emitted light, and computer assisted automated means configured for real-time storing and processing time-lapse recorded data, and displaying said processed data, and wherein the fluidic system further comprises computer assisted automated means configured for driving the high-precision pump and the flow-through pump.

It is part of the invention that using the automated analytical system of the invention for optically measuring complex formation under flow at physiological shear rate only requires a very minimal training and level of skills. It is preferred that using the automated analytical system of the invention equals touching a single button for starting and executing a measurement with the method according to the invention. Of course, it is equally preferred that the automated analytical system of the invention is for example equipped with a user interface comprising a touch screen.

The computer assisted means according to the invention comprise at least two cores with two threads per core, or comprise at least four cores, for fast and

simultaneously recording images and processing recorded images.

Figures 1 -3 provide an overview of typical examples of embodiments of the invention, i.e. the parts of an automated analytical system of the invention applicable for use in the method of the invention, i.e. flow cells according to the invention, and an imaging assembly according to the invention, and fluidic systems according to the invention. These figures also provide exemplified flow cells 4 according to the invention, such as the flow cells 4 comprised by the automated analytical system according to the invention. Figure 2 provides a detailed scheme of the imaging assembly part of the automated analytical system of the invention.

A further aspect of the invention is flow cell 4, 4' for use in the automated analytical system according to the invention, comprising at least one flow channel 43, preferably two flow channels 43, 43', said flow channel comprising at least one reaction zone 45 located at a portion of the flow cell that is transparent for light, and said reaction zone comprising an immobilized binding partner, and said flow channel having an inlet 42, 42' positioned at an angle of 5-90° relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the at least one reaction zone in the flow channel, preferably at an angle of 10° to 20°, more preferably 1 1 ° to 12°, most preferably about 1 1 °, and an outlet 44 positioned at about the same angle as the angle of the inlet relative to the longitudinal dimension (length) and/or the transversal dimension (width) of the reaction zone, and having a cross-sectional area of 0.075 mm ² to 0.30 mm ² , preferably with a transversal width of about 2 mm or about 3 mm and a height of about 50 micrometer, with the reaction zone having a surface area of 1 .25 mm ² to 15 mm ² , preferably with a longitudinal dimension (length) of 0.8 mm to 3 mm and a transversal dimension (width) of 2 mm to 3 mm.

The angle between the inlet (and outlet) and the surface of reaction zone provides for an optimal flow of the aqueous sample. By applying a flow cell with an inlet and an outlet with an angle of about 10° to 20°, preferably about 1 1 ° according to the invention, provides the advantage of a constant viscosity of the aqueous sample flowing through the flow channel, over the whole length of the flow channel. A further advantage of these selected angles according to the invention is the minimized collision and interaction of mobile binding partner with the walls of the inlet and outlet of the flow channel and the wall of the flow channel, when entering and exiting the flow channel. For example, when the mobile binding partner is a platelet, minimalized collision and interaction with the inlet and the flow channel surface prevents pre-activation of the platelet, before it is activated and binding to immobilized binding partner of the reaction zone in the flow channel.

The cross-sectional area of the flow channel of the invention is selected for the optimized flow conditions achieved with such area, according to the invention. The selected cross-sectional area allows for a desired physiological shear rate in the flow channel with a minimized volume of aqueous sample required to achieve the desired shear rate. Of course, minimizing aqueous sample volume is beneficial for, for example, a patient awaiting test results for personalizing the therapy to be selected, when the aqueous sample is whole blood. The combination of flow and cross-sectional area is also selected such that the width of the channel is optimal for laminal flow with a homogeneous shear rate along the full width of the flow channel.

Preferably, the reaction zone comprising the immobilized binding partner is rectangular with a longitudinal dimension (length) of about 0.8 mm to about 3 mm, according to the invention. Applying rectangular reaction zones has several advantages according to the invention. First, rectangular reaction zones are easily provided in a highly reproducible manner, for example using a pipetting robot. Second, the longitudinal dimension or length of the reaction zones is easily fine-tunable with regard to the minimal length required in order to allow for complex formation with a mobile binding partner. For example, platelets in whole blood require a minimal reaction zone length comprising collagen of about 0.8 mm, when the flow of the aqueous sample, i.e. the blood, is such that physiological shear rate is achieved in the flow channel. This minimal longitudinal length is for example for platelets determined by a minimal path length for contacting an activating immobilized binding partner while still (partly) in the aqueous solution, and a subsequent minimal path length for adhering to the immobilized binding partner (complex formation).

Preferably, according to the invention, a reaction zone comprises about 1 μg to 20 μg immobilized binding partner, more preferably about 1 .5 μg to 15 μg, most preferably about 1.5, 3, 6, 9, 12, 15 ng.

As said before, the inventors surprisingly found a combination of features related to the process of complex formation under flow and physiological shear rates within a sufficiently short time-frame, i.e. within 30 minutes, preferably within 4 to 6 minutes, and related to acquiring high-quality time-lapse recorded signal data combined with time- efficient data processing, addressing the aforementioned limitations in the art. Amongst the features that contribute to the sufficiently quick complex formation of sufficient quality and extent in order to allow acquiring data of sufficiently high quality and intensity, the size of the surface area of the reaction zone 45 makes a contribution. The inventors found that typically reaction zones 45 with a surface area of 1.25 mm ² to 10 mm ² , preferably about 1 .5 to 6 mm ² are suitable in the method of the invention. Moreover, the inventors found that preferably, reaction zones 45 are rectangular in shape, with a longitudinal dimension (length) of about 2 mm and a transversal dimension (width) of about the width of the flow channel 43, preferably about 2 mm or about 3 mm. Such reaction zones 45 of the invention expose immobilized binding partner in a manner typically suitable for efficient and timely complex formation with mobile binding partner in the method according to the invention. These sizes and shapes of the reaction zone 45 in the flow cell 4 of the invention are particular suitable for the method according to the invention in connection with a flow channel 43 having a cross-sectional area of 0.075 mm ² to 0.30 mm ² , preferably with a width of about 2 mm or about 3 mm and a depth of about 50 micrometer, according to the invention. Particularly these combinations of cross-sectional area and surface area and shape of the reaction zone 45 enable the beneficial features of the method of the invention with regard to the short time line to relevant data acquisition due to sufficiently fast complex formation under flow and physiological shear rate.

As said, the invention provides for a balanced combination of an imaging assembly, a flow cell and a fluidic system, enabling the simultaneous complex formation under flow at physiological shear rate and recording, storing and processing of images. With the microscope objective lens, for example magnifying 60x and for example a CMOS sensor, for example 2 megapixel images are recorded, with an image showing a surface area of the reaction zone of 160 x 200 μηι ² . In this example, the resolution is 144 pixels per squared micrometer. This resolution suffices for retrieving the relevant parameters upon processing the recorded images for determining the haemostatic status of the patient. This exemplified image size for example allows for sufficiently fast image data storage and processing, given a processor speed of a selected computer assisted means for real-time storing and processing time-lapse recorded image data. Of course, applying a processor which has a higher speed allows for higher resolution images, for example 8 megapixel images. However, for example the resolution of 144 pixels per squared micrometer provides sufficiently high quality input images for processing and

subsequently for providing test results.

One of the important aims of the present invention was the provision of a method for optically measuring complex formation under flow at physiological shear rate between a mobile binding partner and an immobilized binding partner, which would require a minimal level of steps before test results are obtainable, and which would require as much low-tech sample handling as possible. One way to facilitate this aim, is providing a flow cell 4" according to the invention for use in the method according to the invention, in which the flow cell 4" is provided with a sample holder 2'. A preferred examples of such a flow cell 4" comprising a sample holder is provided in Figure 1 C.

Thus, preferably, the flow cell 4" according to the invention comprises a sample holder 2' in connection with the flow channel 43, 43', with the inlet 42, 42' for the aqueous sample in connection with a connector 3a for connecting the flow cell to the flow-through micro-pump 5, 15 of the fluidic system according to the invention, and with the outlet 44, 44' connected to the high-precision pump 1 '.

Importantly, according to the invention, the flow cell 4 of the invention comprising at least one reaction zone 45 with immobilized binding partner, is readily conserved for at least six months at frozen conditions, e.g. at -20°c to -30°C or at about -70° to -85°C, preferably at about -20°C or at about -80°C according to the invention. In addition, the at least one reaction zone 45 with immobilized binding partner, is also readily conserved for at least 6 months when the immobilized binding partner is lyophilized once immobilized at the reaction zone 45. Typically, the flow cell comprising a reaction zone with immobilized binding partner is stored frozen. Preferably, storage conditions are under liquid nitrogen or liquid helium. Storing flow cells frozen according to the invention provides the opportunity to fabric large quantities of flow cells with reaction zones comprising immobilized binding partner in a single batch, providing flow cells with constant specifications. Furthermore, the risk for expiry of the reaction zone of the flow cells before application of the flow cells in stock is reduced. See for a typical embodiment of the invention Figure 4 and Example 7.

Typical materials and surfaces suitable for application as a reaction zone 45 according to the invention are conventional glass and polydimethylsiloxane (PDMS). Of course, other materials routinely applied in the field of complex formation between a mobile binding partner in an aqueous sample and an immobilized binding partner are equally applicable for use as a reaction zone 45 in the flow cell 4 of the invention and in the method according to the invention. Further examples of materials applicable for use as a reaction zone 45 are polymers routinely used in ELISA based assays, mica, saffire, and the like.

The top part 40 of a flow cell 4 according to the invention (See for an example of a flow cell 4, Figure 1 ) is made of glass, preferably PDMS, or other (transparent) materials such as polycarbonate, polyethylene (PE), polystyrene (PS).

The method of the invention has been proven to be exceptionally suitable for use in the assessment of haemostatic conditions and coagulation status of a subject, preferably a human subject. Preferably, the use of the method according to the invention provides the relevant information on the activation of platelets, formation of a thrombus, platelet-based coagulation with platelets of a patient. More preferably, the use of the method according to the invention provides test results relating to the platelet aggregation status, platelet adhesion status and/or platelet-fibrin interaction status in a whole blood sample of a patient. Use of the method according to the invention is typically suitable for providing parameter values regarding platelet aggregation, thrombus size, granule secretion, fibrinogen binding, clotting time and maximum lysis. These parameters provide guidance to for example the ones responsible for selecting at least one adequate therapy for a patient suffering from (severe, acute) bleeding or from (acute) thrombosis, for example in the perioperative or postoperative context, for example during or after surgery in the operation theater. Typically preferred is the use of the analytical system according to the invention and a flow cell 4 according to the invention for measuring haemostasis with the method according to the invention by measuring at least one parameter selected from platelet deposition (thrombus surface area), thrombus build up, number of thrombi per surface area of immobilized binding partner, multilayer, P-selectin expression, phosphatidylserine exposure, fibrinogen binding, platelet adhesion under non-coagulating conditions, and/or by measuring platelet deposition (thrombus surface area), thrombus build-up, time-to- fibrin formation, fibrin formation under coagulating conditions, and/or by measuring fibrinolysis under coagulating conditions, platelet-fibrin interaction, platelet aggregation, platelet adhesion, annexin A5 binding, granule secretion, formation of thrombin, platelet accumulation, thrombus volume, thrombus stability, immobilized platelet contraction, stenosis, granule secretion, clotting time, maximum fibrinolysis, platelet-based

coagulation, or combinations thereof, preferably selected from platelet activation, platelet adhesion, thrombus formation, platelet-dependent fibrin formation under coagulating conditions, platelet-based coagulation, wherein the aqueous sample is whole blood.

Of course, now that the inventors provide a method for providing relevant detailed information on complex formation in a meaningful short time-frame with regard to e.g. a bleeding patient or a patient at risk of thrombosis in the operation theater in need of receiving acute therapy to be selected, the method is also applicable for various different purposes. For example, the method of the invention is for bed-side monitoring of haemostasis conditions and coagulation status, aiding in improved care. Also the influence of antithrombotic agents on haemostasis can be assessed under flow conditions at physiological shear rate, with the method, the analytical system and the flow cell 4 of the invention. In fact, the invention provides for use of the analytical system and a flow cell 4 according to the invention in drug-screening programs aimed at determining the influence of tested drugs or druggable compounds on the one or more parameters attributing to haemostasis and coagulation status in a subject, preferably a human subject. Furthermore, the analytical system and a flow cell 4 according to the invention are particularly suitable for use in screening aqueous samples, such as blood samples, for defects related to impairment of haemostasis in the subjects. Preferably, these further applicable uses of the method according to the invention and of the analytical system and a flow cell 4 according to the invention are with whole blood, although other aqueous samples comprising a suitable binding partner are also applicable. Other aqueous samples comprising a mobile binding partner applicable for use in the method according to the invention are platelet-rich plasma, platelet free plasma, serum, isolated blood components, such as leukocytes, neutrophils, progenitor cells.

Thus, use of the analytical system according to the invention and a flow cell 4 according to the invention is preferred for determining the influence of a protein mutation or a protein modification and/or for determining the influence of a cell defect in the mobile binding partner or in the immobilized binding partner and/or for determining the influence of the concentration of the mobile binding partner or the immobilized binding partner, on complex formation under flow at physiological shear rate between the mobile binding partner in an aqueous sample and an immobilized binding partner.

Thus, in a preferred use of the analytical system and a flow cell 4 according to the invention, the aqueous sample is whole blood, and the whole blood is selected from a subject who is healthy subject, or who is a patient:

o suffering from von Willebrand factor disease, Bernard Soulier

syndrome, a platelet function disorder, thrombocytopenia before and after transfusion, fibrinogen deficiency, an allb33 inhibitor, a coagulation factor deficiency, severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May- Hegglin anomaly, dense granule defect, grey platelet syndrome, SCID, haemophilia B;

o with risk for cardiovascular disease, a vascular disease, an inflammatory disease, a known platelet defect, an unknown platelet defect, a haemophilia, a cancer, diabetes, obesity, a congenital disease such as Noonan;

o who underwent chemotherapy or any other related therapy;

o receiving antiplatelet therapy, receiving a coagulation factor such as Factor VIII and von Willebrand factor;

o who receives anti-thrombotic therapy;

o who receives tyrosine kinase inhibitor therapy;

o in the perioperative setting;

o who undergoes or underwent cardiac operation;

o who undergoes or underwent a cardiopulmonary bypass; o who undergoes or underwent vascular operation;

o who undergoes or underwent a surgery;

o who undergoes or underwent a bone marrow transplantation, or combinations thereof. Preferably, a whole blood sample is measured, wherein the whole blood sample is derived from a patient suffering from cancer, thrombocytopenia before and after transfusion, a patient who receives tyrosine kinase inhibitor therapy, a SCID patient, a haemophilia B patient, or from a patient that underwent a bone marrow transplantation. Preferred embodiments of the invention in this regard are provided in Figures 5-7 and Examples 8, 12, 15 and 16.

With the method of the invention, it is particularly suitable to screen for patients and select patients suffering from any of the diseases or health issues related to von Willebrand factor disease, Bernard Soulier syndrome, clopidogrel therapy, platelet function disorders, fibrinogen deficiency, allb33 inhibitors, coagulation factor deficiency, hyperfibrinolysis, heparin / hirudin effects, or combinations thereof.

Optionally, the analytical system according to the invention and a flow cell 4 according to the invention is used for determining the influence of a compound on protein- protein-, protein-cell-, cell-cell interactions.

Optionally, the analytical system according to the invention and a flow cell 4 according to the invention is used for determining the influence of a gene mutation or a gene defect, protein mutation or protein modification, or cell defect on protein-protein-, protein-cell-, cell-cell interactions.

Now that the invention provides for a fast method for assessing haemostasis parameters and coagulation status, requiring a minimal level of skills of the operator using the method, the method is also equally suitable for screening purposes, e.g. high- throughput screening purposes. For example, series of druggable compounds can be screened routinely, or populations can be screened for hereditary or acquired defects at the DNA level and/or at the protein level of subjects, influencing haemostasis in the subjects, and test results are for example compared to reference values in a database. For these applications and for the general use of the method and the apparatus of the invention, the invention also provides for a kit comprising the essential elements for routine use of the method according to the invention.

Thus, it is an important aspect of the invention that a kit of parts is provided comprising:

a) a flow cell 4, 4', 4" according to the invention for use in the automated analytical system according to the invention;

b) at least one holder with a further solution, selected from Ca ²⁺ /Mg ²⁺ solution, physiological salt solution, buffer solution, wash buffer, rinse buffer, solution comprising a fluorescent label; c) optionally a holder with at least one light emitting label according to the invention; and

d) a sample holder 2, wherein the sample holder is a syringe for blood draw, said syringe pre-filled with citrate and optionally pre-filled with at least one light emitting label according to the invention and with PPACK,

for use in a method according to the invention.

Preferably, the kit of parts according to the invention comprises a disposable flow cell 4 and disposable holders. Thus, the kit of parts according to the invention comprises the disposables required for applying the method of the invention with the apparatus of the invention.

Moreover, in addition to the aforementioned embodiments of the invention that relate to human health and disease, the method according to the invention is equally applicable for the assessment of complex formation, which complex formation relates to animal health and disease. In this regard, the method of the invention is also applicable as an alternative or as an additive to pre-clinical and clinical animal studies. Such animal studies are for example model studies that mimic a certain complex formation in the human body. Typically, the method of the invention replaces animal studies that are model studies for assessing human coagulation status and haemostatic balance.

Preferably, when the method of the invention is used as an alternative to animal studies, the immobilized binding partner comprises a cell, preferably a cell selected from the aforementioned list of cells selectable as immobilized binding partner. This way, the method of the invention typically replaces current animal models that requiring testing in for example dogs, rabbits, mice, pigs, monkeys, Guinea pigs, etc. Of course, also the use of the method of the invention for replacing animal studies and animal testing is part of the invention. Further, the method of the invention is also preferably used as a standardized research tool with respect to gathering human and animal coagulation data and haemostasis data in the academic setting.

The present invention will be illustrated further by means of the following non- limiting Examples.

EXAMPLES

Applying various modes of the method according to the invention, using an analytical system according to the invention, test results applicable in the clinic were obtained related to: Thrombus formation with whole blood from healthy donors or patients;

Thrombus formation with said whole blood under non-coagulated conditions; and Thrombus formation with said whole blood under coagulating conditions. Example 1

A flow cell according to the invention as part of the analytical system of the invention, suitable for use in the method according to the invention

The illustrative example of a flow cell 4 according to the invention comprising two flow channels 43, 43' is provided in Figure 1A and is constructed from two main parts, the top 40 and bottom part 41. The top part 40 consists of a single component, though may also be a multi-component part. The inlet 42, 42' is positioned at an angle between 5° to 90°, preferably about 1 1 ° related to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface of the reaction zone 45, which allows aqueous sample, e.g. blood to flow through the flow channels 43, 43' under stress-free conditions to prevent e.g. activation of cells or proteinaceous molecules in the aqueous sample comprising the mobile binding partner such as for example blood coagulation factors and/or platelets. Similarly and for similar reasons, the outlet 44, 44' is positioned at an angle between 5° to 90°, preferably about 1 1 ° related to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface of the reaction zone 45. Most preferable, the two angles are the same or similar. The bottom part 41 of the flow cell 4 is made of glass or of any optically transparent plastic or other material known in the art and comprises the at least one reaction zone 45 comprising the immobilized binding partner according to the invention. Most preferably, the bottom part 41 and the top part 40 of the flow cell 4 is made of a material that is at least transparent at the location of the reaction zone(s).

Furthermore, this material does not or only to a very tiny extent have an influence on complex formation.

Remark: for clarity, the top part 40 and the bottom part 41 are depicted with a spacing in between, indicated with the dashed lines. In the flow cell 4 according to the invention, the top part 40 and the bottom part 41 are in intimate contact. For example, the parts are glued together, or fixed to each other by any other means known in the art. This flow cell 4 is for example suitable as a part in the analytical system according to the invention, as depicted in Figure 3B.

In Figure 1 B, a flow cell 4' is depicted, which flow cell comprises a single flow channel 43. This flow cell 4' is suitable as a part in the analytical system according to the invention, as depicted in Figure 3A. In Figure 1 C, a flow cell 4" is depicted, having a sample holder 2' in connection with the inlet 42 of the at least one flow channel 43 of the flow cell. According to the invention, flow cell 4" is applied in the automated analytical system according to the invention for which a preferred embodiment is provided as Figure 3C.

Notably, the inlet and the outlet of the flow channel of the flow cell according to the invention are also applicable for use as the outlet and the inlet of the flow channel, respectively, according to the invention. Of course, as said before, when multiple reaction zones are provided in the flow channel, the order in which mobile binding partner contacts subsequent immobilized binding partners may have an influence on test result outcomes of measurement.

Example 2

An imaging assembly of the automated analytical system for optically measuring complex formation under flow at physiological shear rate between a mobile binding partner and an immobilized binding partner, according to the invention.

See Figure 2A.-C. for an imaging assembly 50 as integrated part of an automated analytical system according to the invention.

The top light source 30 is a wide spectrum visible light source that emits light in the visible range (300 nm to 750 nm). Preferably, the top light source 30 is a diffuse white LED, suitable for brightfield transmission recordings, such as a non-modulated LED or a pulsed modulated LED. Light passes through the flow-cell 4' of the analytical system according to the invention, in which the complex formation occurs, e.g. in which thrombus formation is assessed, and the light is passed on to the (commercially available) microscope objective lens 31. This lens is preferably an oil lens, such as the Olympos 60x magnifying oil lens. Magnification of 40x also suffices, and also lenses with other magnifications are suitable. Other suitable lenses are for example water lenses or air lenses known in the art. When the light passed through the lens 31 , the magnified light is reflected on a mirror 32, which is preferably a high-reflectance mirror. Preferably, this mirror minimally reflects 99%, and preferably this mirror is a silver mirror, or the like. The bottom light source 33 is mainly used for emission of narrow spectrum light for fluorescent excitation of dyes in the range of 390, 465, 488, 549 and 647 nm (all +/- 20 nm). For this, for example either a broad spectrum lamp 33 is applied, preferably a diffuse RGB LED known in the art, or up to four fluorescence lamps 33 each emitting a selected wavelength are applied. Lamp 33 is preferably either a non-modulated LED, or a pulsed modulated LED. This emitted light is reflected onto the flow-cell by a semi-transparent mirror 34 and the high reflectance mirror 32. For a set-up according to the invention, for up to four fluorescent light sources a different mirror is mounted with each filter mirror optimally matching the excitation and emission wavelengths of the fluorescent labels used according to the invention. Preferably, a multi dichroic mirror is applied, capable of filtering for example up to four wavelengths. Of course, also set-ups with mirrors suitable for more than four excitation and emission wavelengths are equally suitable, for the method according to the invention. Alternatively, a wide spectrum visible light is used at position 33, to achieve reflection microscopy. A narrow band filter 35 allows for selectively focusing on the fluorescent label used. The filter 35 is exchangeable and is be matched with the wavelength selected at 33, using a filter-wheel (automated). The filter 35 is an emission filter wheel and preferably comprises 1 to 4 wavelength filters, although it may also comprise more than 4 wavelength filters. The emitted and transmitted light is projected on the imaging sensor 37 using an imaging lens 36. Signal data is time lapse recorded and stored on a data carrier coupled to a computer device, e.g. a(n external) hard disk (not shown). The imaging sensor 37 is preferably a CCD sensor known in the art, or a CMOS sensor known in the art, such as a Sony IMX-174 CMOS sensor or a Sony IMX-249 CMOS sensor. Preferably, the sensor is equipped with a global shutter, although a sensor with a non-global shutter is also suitable for use in the method of the invention, if lamp 30 and lamp 33 are non-modulated LEDs. The shutter of the sensor according to the invention preferably has a shutter time of about 10 milliseconds (ms) to about 200 ms.

In Figure 2A., trans-illumination of the flow cell 4' is shown. The top light source 30 is emitting light and illuminates the reaction zone 45 in the flow cell 4' with diffuse light. Transmitted light is captured by the objective lens 31 positioned below the flow cell and is forwarded onto the mirror 32. By selecting the suitable semi-transparent mirror 34 a substantial amount of light is passed through and imaged onto the imaging sensor 37. In this configuration there is no filter 35 in use.

In Figure 2B., fluorescence via bottom illumination of the flow cell 4' is outlined, as used for recording fluorescence signal data (images). Now, the top light source 30 is not emitting light. The bottom light 33 is on, the emitted light is reflected by the semi- transparent mirror 34 and passed onto the mirror 32 and then projected in the objective lens 31 and onto the reaction zone in the flow channel. Here the emitted light excites the fluorescent label in the reaction zone 45 in the flow-cell. The fluorescent label emits light signal into any direction, and some of said emitted light is captured by the objective lens 31 , reflected by the mirror 32, passed through the semi-transparent mirror 34. The filter 35 filters out remaining excitation light emitted by the bottom light source 33 and allows the light emitted by the fluorescent label to pass through, which is projected by the lens 36 onto the imaging sensor 37. Subsequently, recorded image data is stored and processed, and optionally compared with reference values for assessed parameters relating to coagulation status and haemostasis, and processed data is displayed in numbers or in graphs.

It has to be understood that the imaging assembly of the analytical system according to the invention also comprises conventional automated means for data storage, data processing and automated display means for at least numerical values and graphical output data, known in the art. For clarity reasons, these means for data storage, data processing and display means are not shown in the Figures 2A and 2B, but these means are an integral part of the analytical system according to the invention.

Preferably, the socket 19 is provided with a thermostat 9, capable of efficiently keeping a flow cell received by a flow cell holder 20 which holder is in intimate contact with the socket, at a predetermined temperature. See Figure 2C. Socket 19 is an integrated part of the imaging assembly according to the invention. The socket is also provided with an xyz stage controller 10, as shown in Figure 2A-C. The controller allows for bringing a reaction zone to be imaged in focus, and for positioning a selected reaction zone in a selected flow channel to be imaged. Example 3

Fluidic system of the automated analytical system of the invention

Figure 3A and 3B and 3C provide examples of preferred embodiments showing three fluidic systems of the automated analytical systems according to the invention, suitable for application in the method according to the invention. For clarity reasons, the imaging assembly of the analytical system according to the invention as outlined in Figure 2A-C is omitted from the Figures 3A-C. As said before, also for clarity reasons, the automated means for data storage, data processing and automated display means are not shown in the Figures 2A and 2B and the Figures 3A-C, but it has to be understood that these means are an integral part of the analytical system according to the invention.

Here, a fluidic system of the invention is exemplified with a system having a flow cell 4' with one flow channel 43 (Figure 3A), and with an analytical system having a flow cell 4 with two flow channels 43, 43' (Figure 3B). In addition, in a further embodiment of the invention, yet a different fluidic system according to the invention is shown (Figure 3C). Of course, analytical systems comprising a fluidic system having a flow cell 4 with a plurality of flow channels 43 of over 2, for example 3 to 6 flow channels are also part of the invention.

Typically and preferably, when the aqueous sample is whole blood, the containers A, B, and C of reservoir 11 and containers D, E, F of reservoir 17 contain the further aqueous solutions recalcification buffer comprising Ca ²⁺ and Mg ²⁺ (A, D), post-perfusing buffer (B, E), and rinse / cleaning buffer (C, F). Also preferred in an embodiment of the invention, at least one container comprises a fluorescent label having affinity for a mobile binding partner. Experiment 4

General experimental conditions with the method of the invention, providing test results applicable in the clinic

The aqueous sample analyzed in the experiments was whole blood from human subjects. Typically, blood was drawn with a conventional vacuum system known in the art, in 3.2% sodium citrate (0.129 M) +/- PPACK (40 μΜ), and kept at 37°C for 5 minutes before start of a measurement according to the method of the invention. For measurements under coagulating conditions, Ca ² 7 Mg ⁺ mix was mixed with the drawn whole blood sample, to obtain as an aqueous sample whole blood which is able to coagulate under fibrin formation.

Application of these aqueous samples provided test results with the method according to the invention, applying an analytical system of the invention, that solved the aforementioned limitations of the current art.

Example 5

Immobilization of the immobilized binding partner

Immobilized binding partner was immobilized in a reaction zone 45 by applying either of two techniques, as depicted below.

Immobilized binding partner having a circular surface area

A circular surface area of an immobilized binding partner was achieved by manual pipetting or by using a robot, on coverslips which are degreased with 2 M HCI in 50% ethanol. Volumes of 0.5 μΙ were for example pipetted on a coverslip. When a robot is used a MicroGrid II robot is used using a printing pin of >700 μηη. Immobilization methods ('coating') result in a circular surface area of 1 .5 mm ² . For both methods coated coverslips are kept in a humid chamber with >50% humidity for 60 minutes. Blocking of coverslips is performed with 1 % BSA (in HEPES, pH 7.45). After blocking, coverslips are rinsed with saline and mounted on the flow cell. When applying a volume of 2 μΙ of binding partner onto the coverslip, a circular surface area with a diameter of 2 mm is obtained. Typical immobilized binding partners according to the invention are listed in Table 1.

Preferably, according to the invention, a reaction zone comprises about 1 μg to 20 μg immobilized binding partner, more preferably about 1 .5 μg to 15 μg, most preferably about 1.5, 3, 6, 9, 12, 15 μg.

Immobilized binding partner having a rectangular surface area

TABLE 1

Experimental conditions for application of the method according to the invention with whole blood being the aqueous sample, the whole blood applied under coagulating conditions and under non-coagulating conditions during measurements of complex formation.

Immobilized binding partners as well as applied shear rates in an exemplified

measurement with the method of the invention are listed.

Aqueous

Immobilized binding partner sample

conditions

Coating Source Concentration Diluent n.c / c Shear rate is ^"1 )

Collagen (l/lll) Bovine 50 or 100 Collagen n.c 150 / μg mL buffer 1 .600

Von Willebrand Human 50 μg/mL + mQ +PBS n.c 1 .600 factor + laminin plasma + 100 g/mL

placenta

Von Willebrand Human 50 μg/mL + mQ + saline n.c 1 .600 factor + plasma 250 g/mL

fibrinogen

Von Willebrand Human 50 μg/mL + mQ c 1 .600 factor + plasma + 250 g/mL

rhodocytin snake venom

Collagen + Bovine + 50 μg/mL + Collagen c 150 / tissue factor recombinant 500 pM buffer + 1 .000

(E.coli) HEPES buffer

Collagen + Bovine + 50 μg/mL Collagen 150 / activated human + 5 nM buffer + 1 .000 protein C plasma HEPES buffer

Rectangular surface areas of immobilized binding partner were obtained by so-called 'stream coating'. Coverslips were selected as the bottom part 41 of the flow cell 4 according to the invention, and coverslips are degreased with 2 M HCI in 50% ethanol. Ten μΙ of solution comprising the binding partner to be immobilized in a reaction zone 43 was perfused (10 μΙ volume) through a coating channel perpendicular to the longitudinal direction of the flow channel 43. This assembly was kept in a humid chamber with >50% humidity for 60 minutes. After 60 minutes the flow channel 43 comprising the immobilized binding partner was perfused with saline. Blocking of coverslips is performed with 1 % BSA (in HEPES, pH 7.45). After blocking, the bottom part 41 coverslips of the flow cell 4 are rinsed with saline and mounted on the top part 40 of the flow cell 4. Overall this way of coating results in reaction zones 45 of rectangular spots of 6 mm ² , when a flow channel 43 with a width of 3 mm is applied. Typical immobilized binding partners according to the invention are listed in Table 1 .

In Table 1 , an overview of experiments is provided, which provides examples of combinations of immobilized binding partners and aqueous samples comprising the mobile binding partner and applied shear rates, that allowed for the provision of at least one test result suitable for use in the clinic, when applied in the method according to the invention. With the combinations of immobilized binding partner, surface areas of the immobilized binding partner and applied shear rates, imaging data is recorded and processed in a time short enough to be of clinical relevance, i.e. within about 6 minutes from the start of the measurements using the method of the invention with the analytical system according to the invention. Abbreviations: n.c = non-coagulating conditions; c = coagulating conditions; mQ = ultrapure water For the experiments outlined in Table 1 , immobilized binding partner having a circular surface area as well as having a rectangular surface area were applied. The experiments depicted in Table 1 were performed with the method according to the invention, using flow cells 4 constructed from either hard plastic material, or from soft PDMS material. Typically, flow channel 43 dimensions were either 2 mm or 3 mm (width) and 50 μηη (height). The flow cell 4 inlet 42 and outlet 44 have an angle of 1 1 ° relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zone 45, unless indicated otherwise. Example 6

Assessing time lines required to obtain test results with the method of the invention, using an analytical system according to the invention

In order to demonstrate that by applying the method of the invention using an analytical system according to the invention, test results are automatically provided within 6 minutes, the following examples are provided.

An automated analytical system according to the invention and according to Figure 3A was applied in the method of the invention. The flow cell 4 of the analytical system thus comprised a single flow channel 43. In Table 2, test results are provided with collagen as the immobilized binding partner and either whole blood under non-coagulating conditions, or whole blood under coagulating conditions, as the aqueous sample. The test results show that when the method of the invention is run using an automated analytical system according to Figure 3B, measuring blood under non-coagulating conditions and whole blood under coagulating conditions in parallel in the two flow channels 43, all test results are available within 6 minutes. Table 3 provides an overview of test results that were obtained with these aqueous samples.

In a typical example of the automated method of the invention, at t = 0 Ca ² 7 Mg ²⁺ was mixed with an anti-coagulated whole blood sample, for complex formation under coagulating conditions. The automated analytical system according to the invention automatically rinsed the complex formed in a flow channel wherein complex was formed under coagulating conditions, and automatically rinsed the complex formed in a flow channel wherein complex was formed under non-coagulating conditions. After rinsing, buffer comprising fluorescent label was flown through the flow channels, and

subsequently, a cleaning step comprising flowing cleaning buffer through the flow channels was applied. Subsequently, fluorescence was recorded. Test results for the whole blood sample applied under coagulating conditions were obtained within 6 minutes. Test results for the whole blood sample applied under non-coagulating conditions were obtained after 4 minutes of blood perfusion through the flow channel, followed 2 minutes of rinsing buffer / label / cleaning buffer perfusion, thus also within 6 minutes. Test results were automatically made available as numerical values, as a mean value +/- 2 SD, and as color images. Furthermore, test results were automatically compared with reference test results acquired from whole blood samples of healthy control subjects. All blood samples were from human subjects.

Test results obtained this way were:

Non-coagulating conditions, assessing measures for the rate of platelet adhesion, aggregation and activation

Platelet adhesion in time (area under the curve (AUC) + slope) to a combination of platelet receptors / via different pathways.

Integrated feature size (See below) and fibrinogen binding (measure for capability of platelets to aggregate).

P-selectin expression (measure for platelet activation)

Phosphatidylserine (PS) exposure (measure for procoagulant activity in percentage surface area coverage (% SAC)) Integrated feature size:

The integrated feature size was determined as a parameter, taking into account a proportional contribution of large and small thrombi on microspots. It was defined as described in De Witt et al., 2014, page 12, left column, "Quantitative analysis of recorded images", which is incorporated by reference.

Coagulating conditions, assessing measures for the amount of platelet-dependent coagulation

Platelet adhesion to the immobilized binding partner and fibrin formation in time (time-to-fibrin formation + AUC + slope).

Table 2

Test conditions and time required for obtaining test results with whole blood samples and collagen in the method of the invention, using an analytical system according to the invention Experimental Non-coagulating

Coagulating conditions Parameters conditions

Flow 4.5 mL/h 4.5 mL/h or 0.675 mL/h

Shear rate 1 .000 s ^"1 1 .000/s or 150 s ^"1

Time of experiment 6 minutes 6 minutes

Time lapse data recording 4 minutes 6 minutes

End-point measurement 1 minute 0 minutes

AUC = area under the curve

Example 7

Effect of storage of immobilized binding partner under frozen conditions and after

Ivophilization Coverslips comprising immobilized binding partner in reaction zones 45 are kept at -20°C for 9 days. Whole blood was the aqueous sample, platelets were the mobile binding partner. The immobilized binding partner was as indicated below. Thrombus formation was measured prior to freezing and after freezing, with the method according to the invention.

Blood collection. Blood was drawn in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 μΜ PPACK and Ca ² 7 Mg ²⁺ mix (3.75 mM/7.5 mM).

Microspotting. A spot of collagen III and von Willebrand factor (vWF)-fibrinogen (0.5 μΙ_) was applied on a degreased glass coverslip (Table 1 ). Relative to the direction of the flow of the aqueous sample through the flow channel 43, in a first reaction zone 45 vWF- fibrinogen was immobilized, and in a second reaction zone 45 collagen III was

immobilized. Surface areas were about 1 .3 - 1.5 mm ² . Spots were blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the binding partners. After blocking the coverslip (bottom part 41 of the flow cell 4) was mounted on the top part 40 of the flow cell 4, according to the invention.

Type of flow cell. Immobilized binding partners were applied in flow cells 4 made of hard plastic material with flow channel 43 dimensions of 3 mm (width) and 50 μηη (height). Inlet 42 and outlet 44 of the flow channel 43 make an angle of 1 1 ° relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.

Perfusion experiment with the method of the invention. Performed at room

temperature. Recalcified whole blood samples (aqueous sample) were put in a syringe (container 2) rinsed with rinse buffer (further aqueous solution) and connected to the flow cell 4. Blood with platelets (mobile binding partner) was perfused for 4 minutes at 1 .600 s ^" Imaging was performed after 1 minute rinsing with rinse buffer in order to discard red blood cells. End point measurements (brightfield) were performed for determining platelet area coverage (Figure 4). Figure 4 indicates that different immobilized binding partners acting as the adhesive surface for complex formation with platelets result in rather similar amounts of surface area coverage (SAC) of platelet and thrombi before and after freezing (day 1 , before freezing, and day 9, after freezing + thawing, respectively). Mean ± SD representatives of 1 blood donor are depicted in Figure 4. These results show that various immobilized binding partners on a reaction zone 45 are stored frozen without substantial loss of complex-forming capacity. Example 8

Identification of haemostatic status in patients

With the method of the invention, applying an automated analytical system according to the invention and applying a flow cell 4 according to the invention, the following aqueous samples were successfully analyzed for the presence of coagulation abnormalities and/or abnormalities related to thrombus formation:

whole blood samples under coagulating conditions as well as under non-coagulating conditions of patients with:

a) cardiovascular risk;

b) known platelet defect;

c) unknown platelet defect;

d) congenital disease such as Noonan;

e) cancer disease and chemotherapy;

f) receiving antiplatelet therapy;

g) receiving coagulation factors (e.g. factor VIII, von Willebrand factor);

h) thrombocytopenia before and after transfusion; and

i) other (non-)related diseases.

Example 9

Use of the method of the invention with a flow cell comprising several reaction zones in the flow channel

The following experiment demonstrates the use of the method of the invention for assessing complex formation between platelets from whole blood and immobilized binding partners, immobilized in a series of reaction zones 45 in the flow channel 43.

Measurements were performed under non-coagulating conditions.

Blood collection. Blood was drawn from a healthy donor in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 μΜ PPACK and Ca ² 7 Mg ²⁺ mix (3.75 mM/7.5 mM).

Microspotting. A spot of collagen type I, collagen III and vWF-fibrinogen (0.5 μΙ_) was applied on a degreased glass coverslip (Table 4). Spots were coated in series in the direction of the flow going from vWF-fibrinogen, collagen III to collagen I, respectively. Surface areas of the reaction zones were in the range from 1 .2 to 1 .5 mm ² . Reaction zones were blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein, i.e. the immobilized binding partner. After blocking the coverslip (bottom part 41 )was mounted on the top part 40 of a flow cell 4. Type of flow cell. Coatings were applied in flow cells 4 constructed out of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 μηη height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.

Perfusion experiment. Performed at room temperature. Recalcified whole blood samples were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 4 minutes at 1 .600/s. Imaging was performed after 1 minutes rinsing with rinse buffer in order to discard red blood cells. End point measurements (brightfield) were performed for assessing platelet area coverage (Table 4). Table 4 indicates that different adhesive surfaces (immobilized binding partners) give different amounts of surface area coverage (SAC) of platelet (mobile binding partner) and thrombi.

Table 4. Mean SAC (%) ± SEM of 2 experiments

Example 10.

Experimental effect of co-coating (rectangular reaction zones: 1 x2 mm)

Blood collection (non-coagulating conditions). Blood was drawn from a healthy donor in Vacutainer tubes containing 3.2% of sodium citrate.

Stream spotting. A stream coating was made with collagen type I, collagen type III or vWF-fibrinogen (immobilized binding partners) on a degreased coverslip (Table 5) using a volume of 2 μΙ_. Spots were coated in the same order as described in Example 9 with size of 1 mm (length) x 2 mm (width).

Type of flow cell. Coatings were applied in flow cells 4 constructed out of PDMS material with flow channel 43 dimensions of 2 mm width and 50 μηη height. Inlet 42 and outlet 44 have an angle of 90°, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones.

Perfusion experiment. Performed at room temperature, with the method of the invention, applying an automated analytical system of the invention. Anticoagulated whole blood samples (aqueous samples) were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 4 minutes at a shear rate of 1 .600 s ^' Imaging recording was performed after 1 minutes rinsing with rinse buffer in order to discard the red blood cells. End point measurements (brightfield) were performed for assessing platelet area coverage (Table 5) and Annexin-A5 binding was determined as a measure of the procoagulant activity. Table 5 indicates that different adhesive surfaces (immobilized binding partners) result in different amounts of surface area coverage (SAC) of platelet and thrombi. These data show that stream coating is adequate to determine more than one thrombus formation marker on different platelet adhesive surfaces as the immobilized binding partners.

Table 5. Mean surface area coverage (SAC) (%) ± SEM of 2 experiments

Example 11.

Experimental effect of co-coating (rectangular reaction zones: 2x3 mm (length x width)) Blood collection. Blood (aqueous sample) was drawn from a healthy donor in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 μΜ PPACK and Ca ² 7 Mg ²⁺ mix (3.75 mM/7.5 mM).

Microspotting. A spot of collagen type I (0.5 μΙ_) was applied on a degreased glass coverslip (Table 1 ). Spot size (reaction zone) was 2x3 mm (longitudinal dimension or length x transversal dimension or width). Spots of immobilized binding partner were blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein (binding partner to be immobilized). After blocking the coverslip (bottom part 41 ) was mounted on the top part 40 of a flow cell 4.

Type of flow cell. Coatings (immobilized binding partners) were applied in flow cells 4 constructed of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 μηη height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zone 45. Perfusion experiment. Performed at room temperature. Recalcified whole blood samples were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 4 minutes at a shear rate of 1.600 s ^' Image recording was performed after 1 minutes rinsing with rinse buffer in order discard the red blood cells. End point measurements (brightfield + DiOC6 fluorescence) were performed concerning platelet area coverage (Table 6). Table 6 shows that there is sufficient thrombus formation on spots (reaction zones) with a rectangular spot size of 2 mm (longitudinal dimension or length) x 3 mm (transversal dimension or width). Table 7. Representative SAC (%) of a single experiment

Example 12.

Thrombus formation at venous shear rate (effect of bone marrow transplantation)

Blood collection. Blood (aqueous sample) was drawn from a healthy donor and from a patient with severe immunodeficiency syndrome (SCID) after bone marrow

transplantation, in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 μΜ PPACK and Ca ² 7 Mg ²⁺ mix (3.75 mM/7.5 mM).

Microspotting. A spot of collagen type I (0.5 μΙ_) (immobilized binding partner) was applied on a degreased glass coverslip (bottom part 41 of flow cell 4) (Table 3). Spot size had a diameter of 1.25 mm ² . Spots (reaction zone 45) were blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein. After blocking the coverslip (bottom part 41 ) was mounted on the top part 40 of a flow cell 4.

Type of flow cell. Coatings were applied in flow cells 4 constructed of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 μηη height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.

Perfusion experiment. Performed at room temperature. Recalcified whole blood samples (aqueous sample) were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 6 minutes at a shear rate of 150 s ^' Image recording was performed after 1 minutes rinsing with rinse buffer containing fluorescent labels for Annexin-A5 binding (procoagulant activity), fibrinogen binding and P-selectin expression (platelet activation markers) in order to discard the red blood cells. End point measurements (brightfield + fluorescence images) were performed concerning platelet area coverage with respect to brightfield and the various fluorescent markers (Table 8). Table 8 shows that at venous shear rate thrombus formation is decreased with respect to platelet adhesion, platelet activation and procoagulant activity, for the blood sample of the patient, indicating that the bone marrow transplantation did not work properly. This was confirmed by other clinical tests, showing the predictive power of the method of the invention. Table 8. Multiparameter thrombus formation in a patient with SCID (single representative experiment)

Example 13.

Thrombus formation under influence of a drug used in the clinic: tyrosine kinase inhibitor Blood collection. Blood was drawn from a healthy donor in Vacutainer tubes containing 3.2% of sodium citrate. Prior to each experiment blood was recalcified with 40 μΜ PPACK and Ca ² 7 Mg ²⁺ mix (3.2 mM/6.3 mM). A tyrosine kinase inhibitor (TKI), used in the clinical setting as a drug (cancer patients) was added prior to whole blood perfusion.

Microspotting. A spot of collagen type I (2 μΙ_; immobilized binding partner) was applied on a degreased glass coverslip (bottom part 41 of flow cell 4) (Table 3). Spot size

(reaction zone 45) had a diameter of 2 mm. Spots (reaction zones 45) were blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein. After blocking the coverslip (bottom part 41 of flow cell 4)was mounted on the top part 40 of a flow cell 4.

Type of flow cell. Coatings (immobilized binding partner) were applied in flow cells 4 constructed of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 μηη height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones 45.

Perfusion experiment. Performed at room temperature. Recalcified whole blood samples were put in a syringe (container 2) rinsed with rinse buffer and connected to the pump 1 and the flow cell 4. Blood was perfused for 5 minutes at shear rate of 1 .000 s ^' Image recording was performed after 1 minutes rinsing with rinse buffer to discard the red blood cells. End point measurements (brightfield) were performed for assessing platelet area coverage (Figure 5). Figure 5 indicates that at arterial shear rate thrombus formation (complex formation) is decreased due to decreased platelet adhesion under influence of the clinical compound TKI, when added prior to whole blood perfusion, indicating the inhibiting effect of the compound on platelet activation. This test result of the method of the invention using an analytical system of the invention shows one of the many benefits of the invention regarding providing clinicians insight in coagulation status and

haemostasis of patients taking anti-cancer drugs.

Example 14.

Experimental effect of co-coating (circular spots - pipetted), under COAGULATING CONDITIONS

Blood collection. Blood (aqueous sample) was drawn in Vacutainer tubes containing 3.2% of sodium citrate from a healthy donor. During each experiment blood was recalcified Ca ² 7 Mg ²⁺ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio.

Microspotting. A spot of collagen type I, collagen III and laminin (immobilized binding partners) were applied on a degreased glass coverslip (2 μΙ_, giving a diameter of spots of 2 mm) (Table 3). After one hour of incubation, spots (reaction zones 45) were dried using N ₂ and 2 μΙ_ of tissue factor as a second immobilized binding partner was applied on top of the initial coated spots, according to Table 9. Spots were coated in the direction of the flow going from inlet 42 to outlet 44, with laminin, collagen III and collagen I, respectively. Afterwards spots were blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein. After blocking the coverslip (bottom part 41 of the flow cell 4) was mounted on the top part 40 of a flow chamber 4. Type of flow cell. Coatings were applied in flow cells 4 constructed of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 μηι height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal dimension (length) and/or the transversal dimension (width) or the surface area of the reaction zones with immobilized binding partner.

Perfusion experiment. Performed at room temperature. Whole blood samples were put in a syringe (container 2) rinsed with rinse buffer (without heparin) and connected to the pump 1 and the flow channel 43 of a flow cell 4. During each experiment blood was recalcified with Ca ² 7 Mg ²⁺ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio. Blood was perfused at a shear rate of 1.000 s ^' Image recording was performed in real-time (time frames of 15 seconds). Time-lapse measurements (DiOC6 fluorescence signal recording), annexin-A5 binding (coagulation activity) and fibrinogen binding (platelet activation and fibrin formation) were performed during 1 1 minutes of whole blood perfusion (Table 9). This Table 9 shows that a co-coating of collagen with TF as the immobilized binding partners gives similar amounts of thrombus formation but different values with respect to PS exposure and fibrinogen binding, including the effect on time-to-fibrin formation which is lower when collagen is co-coated with TF. Similar results are shown with collagen III and laminin +/- TF in Table 9. These data show that in healthy donors time-to-fibrin formation (TTF) is measured within this flow assay, i.e. the method according to the invention applying an automated analytical system according to the invention, within a timeframe of 6 minutes with clear differences between co-coatings with / without TF. Table 9. The effect of TF co-coating under coagulating conditions (representative data of 1 donor)

Abbreviations: TTF = time-to-fibrin formation, TF = tissue factor, PS = phosphatidylserine

Figure 6 shows the time curve of the three parameters (SAC, PS exposure and fibrinogen binding). When collagen is co-coated with TF, the slope of the curve is steeper indicating that TF triggers coagulation within an even shorter timeframe, shorter than 6 minutes. Example 15.

Experimental effect of co-coating of several immobilized binding partners (circular spots - pipetted) with whole blood sample of a patient with coagulation factor defect, under COAGULATING CONDITIONS

Blood collection. Blood was drawn in Vacutainer tubes containing 3.2% of sodium citrate from a healthy donor or from a patient with hemophilia B (Factor IX defect). During each experiment blood was recalcified with Ca ² 7 Mg ²⁺ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio.

Microspotting. A spot of collagen type I (immobilized binding partner) was applied on a degreased glass coverslip (bottom part 41 of a flow cell 4) (2 μΙ_, giving a diameter of spots of 2 mm) (Table 3). After one hour of incubation, spots were dried using N ₂ and 2 μΙ_ of tissue factor as the second immobilized binding partner was applied on top of the initial coated spots. Afterwards the spot was blocked after 1 hour incubation in a humid chamber (> 50% humidity) to prevent further adhesion of the coated protein. After blocking the coverslip was mounted on a flow chamber.

Type of flow cell. Coatings were applied in flow cells 4 made of hard plastic material with flow channel 43 dimensions of 3 mm width and 50 μηη height. Inlet 42 and outlet 44 have an angle of 1 1 °, relative to the longitudinal orientation of the reaction zone 45.

Perfusion experiment. Performed at room temperature. Whole blood samples were put in a syringe (container 2) rinsed with rinse buffer (without heparin) and connected to the pump 1 and the flow cell 4. During each experiment blood was recalcified by adding Ca ² 7 Mg ²⁺ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio. Blood was perfused for 6 minutes at a shear rate of 1 .000 s ^' Image recording was performed in real-time (time frames of 15 seconds). Time-lapse measurements (DiOC6 fluorescent intensity) and fibrinogen binding (amount of fibrin formation) were performed during 7 minutes of whole blood perfusion. The endpoint measurement on the amount of fibrin formation already confirms the coagulation factor defect in a pre-operative setting (Figure 7). The amount of platelet adhesion, however is similar between the control and the patient (30% and 25% respectively) This shows that, when this patient gets surgery, coagulation factors have to be supplemented in case of bleeding of the patient.

Example 16.

Experimental effect of co-coating immobilized binding partners (circular spots - pipetted) on complex formation of mobile binding partners in whole blood samples of a patient with thrombocytopenia (before and after platelet transfusion), under COAGULATING

CONDITIONS

Blood collection. Blood (aqueous sample) was drawn in Vacutainer tubes containing 3.2% of sodium citrate from a patient with thrombocytopenia before and after platelet transfusion. During each experiment blood was recalcified by adding Ca ² 7 Mg ²⁺ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio.

Microspotting. See Example 15.

Type of flow chamber. See Example 15.

Perfusion experiment. Performed at room temperature. Whole blood samples were put in a syringe (container 2) rinsed with rinse buffer (without heparin) and connected to the pump 1 and the flow cell 4. During each experiment blood was recalcified by adding Ca ² 7 Mg ²⁺ mix (6.3 mM/3.2 mM) at 1 :10 volume ratio. Blood was perfused for 10 minutes at a shear rate of 1 .000 s ^' Platelet deposition (complex formation) was determined at endpoint and the amount of fibrinogen was assessed as a measure of the amount of fibrin present. Platelet surface area coverage and fibrin formation was increased after transfusion (Table 10) which was confirmed by the ratio that is routinely determined in the clinic: the corrected count increment (CCI). This ratio provides an indication of the effect of transfusion. Table 10. Parameters before and after platelet transfusion

Example 17

Platelet aggregation with citrated whole blood and collagen I, without calcium ions, magnesium ions and PPACK

The flow cell according to Figure 1 C was applied in the automated analytical system according to the invention, for measuring surface area coverage of a collagen I (immobilized binding partner) coated reaction zone by platelets (mobile binding partner) with the method of the invention. The aqueous sample was citrated whole blood (human), without calcium ions added, without magnesium ions added (thus, without recalcification), and without PPACK added. The flow cell was made of PDMS or was made of a brittle plastic. The width of the flow channel was 3 mm. Complex was formed for 4 minutes. Test results were provided in these 4 minutes. SAC of the platelets was 31 .4% (SEM 6.3; n = 9) with the PDMS flow cell, SAC was 34% (SEM 1 .8; n = 40) with the brittle plastic flow cell.

These results show that with the method of the invention applying the automated analytical system of the invention, citrated whole blood can be tested as an aqueous sample, without the need for recalcification. These results also show that with the flow cell according to Figure 1 C, test results are obtained within a time span of 4 minutes.

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